0	Molecular Biology of the Cell, otherFeat=[]-->, belongsTo=nota_cab_pie
1	Vol. 20, 5211?5223, December 15, 2009, otherFeat=[]-->, belongsTo=nota_cab_pie
2	Mammalian pre-mRNA 3 End Processing Factor CF Im68, otherFeat=[]-->, belongsTo=title
3	Functions in mRNA Export, otherFeat=[]-->, belongsTo=title
4	Marc-David Ruepp,* Chiara Aringhieri, Silvia Vivarelli, Stefano Cardinale,?, otherFeat=[]-->, belongsTo=title
5	Simona Paro, Daniel Schu?mperli,* and Silvia M.L. Barabino, otherFeat=[]-->, belongsTo=title
6	Department of Biotechnology and Biosciences, University of Milano-Bicocca, I-20126 Milano, Italy; and, otherFeat=[]-->, belongsTo=title
7	*Institute of Cell Biology, University of Bern, CH-3012 Bern, Switzerland, otherFeat=[]-->, belongsTo=title
8	Submitted May 13, 2009; Revised October 14, 2009; Accepted October 19, 2009, otherFeat=[]-->, belongsTo=parrnote
9	Monitoring Editor: Marvin Wickens, otherFeat=[]-->, belongsTo=parrnote
10	Export of mRNA from the nucleus is linked to proper processing and packaging into ribonucleoprotein complexes., otherFeat=[]-->, belongsTo=parr
11	Although several observations indicate a coupling between mRNA 3 end formation and export, it is not known how these, otherFeat=[]-->, belongsTo=parr
12	two processes are mechanistically connected. Here, we show that a subunit of the mammalian pre-mRNA 3 end, otherFeat=[]-->, belongsTo=parr
13	processing complex, CF Im68, stimulates mRNA export. CF Im68 shuttles between the nucleus and the cytoplasm in a, otherFeat=[]-->, belongsTo=parr
14	transcription-dependent manner and interacts with the mRNA export receptor NXF1/TAP. Consistent with the idea that, otherFeat=[]-->, belongsTo=parr
15	CF Im68 may act as a novel adaptor for NXF1/TAP, we show that CF Im68 promotes the export of a reporter mRNA as well, otherFeat=[]-->, belongsTo=parr
16	as of endogenous mRNAs, whereas silencing by RNAi results in the accumulation of mRNAs in the nucleus. Moreover,, otherFeat=[]-->, belongsTo=parr
17	CF Im68 associates with 80S ribosomes but not polysomes, suggesting that it is part of the mRNP that is remodeled in the, otherFeat=[]-->, belongsTo=parr
18	cytoplasm during the initial stages of translation. These results reveal a novel function for the pre-mRNA 3 end, otherFeat=[]-->, belongsTo=parr
19	processing factor CF Im68 in mRNA export., otherFeat=[]-->, belongsTo=parr
20	INTRODUCTION, otherFeat=[]-->, belongsTo=parr
21	The removal of introns by splicing as well as cleavage and, otherFeat=[]-->, belongsTo=parr
22	polyadenylation at the 3 end of RNA polymerase II primary, otherFeat=[]-->, belongsTo=parr
23	transcripts (pre-mRNAs) are usually required before they, otherFeat=[]-->, belongsTo=parr
24	can be exported from the nucleus as mature mRNAs, otherFeat=[]-->, belongsTo=parr
25	(Erkmann and Kutay, 2004). This observation has suggested, otherFeat=[]-->, belongsTo=parr
26	that transport factors interact with the RNA during pre-, otherFeat=[]-->, belongsTo=parr
27	mRNA processing. Indeed, recent discoveries have lent sup-, otherFeat=[]-->, belongsTo=parr
28	port to this hypothesis. The splicing reaction deposits on the, otherFeat=[]-->, belongsTo=parr
29	mRNA a specific subset of proteins called the exon junction, otherFeat=[]-->, belongsTo=parr
30	complex (EJC, for review see Tange et al., 2004). REF, a, otherFeat=[]-->, belongsTo=parr
31	component of the EJC, facilitates mRNA export by inter-, otherFeat=[]-->, belongsTo=parr
32	acting with the mRNA export factor NXF1 (also called TAP,, otherFeat=[]-->, belongsTo=parr
33	for review, see Reed and Hurt, 2002). NXF1 was originally, otherFeat=[]-->, belongsTo=parr
34	identified as the export receptor for type D retroviral RNAs, otherFeat=[]-->, belongsTo=parr
35	that associate with NXF1 through a sequence-specific inter-, otherFeat=[]-->, belongsTo=parr
36	action with the constitutive transport element (CTE). How-, otherFeat=[]-->, belongsTo=parr
37	ever, NXF1 recruitment on cellular mRNAs requires adaptor, otherFeat=[]-->, belongsTo=parr
38	proteins such as Aly/REF (hereafter named REF). In yeast, otherFeat=[]-->, belongsTo=parr
39	Mex67 (the homolog of NXF1) is recruited by Yra1 (homolog, otherFeat=[]-->, belongsTo=parr
40	of REF), which is also essential for the export of poly(A), otherFeat=[]-->, belongsTo=parr
41	RNA in Saccharomyces cerevisiae. In contrast in metazoans,, otherFeat=[]-->, belongsTo=parr
42	REF is dispensable for bulk mRNA export. This raises the, otherFeat=[]-->, belongsTo=parr
43	possibility that multiple and partially redundant adaptor pro-, otherFeat=[]-->, belongsTo=parr
44	teins may be responsible for the recruitment of NXF1. Indeed,, otherFeat=[]-->, belongsTo=parr
45	spliceosomal proteins, including U2AF35 (Zolotukhin et al.,, otherFeat=[]-->, belongsTo=parr
46	2002) and some members of the SR family of splicing factors,, otherFeat=[]-->, belongsTo=parr
47	were shown to interact with NXF1 and act as adaptors for, otherFeat=[]-->, belongsTo=parr
48	NXF1-dependent export of poly(A) mRNAs (Huang and, otherFeat=[]-->, belongsTo=parr
49	Steitz, 2001; Huang et al., 2003; Lai and Tarn, 2004; Hargous, otherFeat=[]-->, belongsTo=parr
50	et al., 2006; Tintaru et al., 2007)., otherFeat=[]-->, belongsTo=parr
51	Several observations have linked 3 end cleavage and, otherFeat=[]-->, belongsTo=parr
52	polyadenylation to mRNA export (for review see Zhao et al.,, otherFeat=[]-->, belongsTo=parr
53	1999). For example, RNA polymerase II reporter transcripts, otherFeat=[]-->, belongsTo=parr
54	lacking a polyadenylation signal are retained in the nucleus, otherFeat=[]-->, belongsTo=parr
55	of yeast cells. Positioning a transcribed poly(A) tract at the, otherFeat=[]-->, belongsTo=parr
56	end of an mRNA by ribozyme cleavage does not result in, otherFeat=[]-->, belongsTo=parr
57	efficient nuclear export, indicating that the 3 end processing, otherFeat=[]-->, belongsTo=parr
58	reaction itself, and not simply the presence of the poly(A), otherFeat=[]-->, belongsTo=parr
59	tail, is required for nuclear export (Huang and Carmichael,, otherFeat=[]-->, belongsTo=parr
60	1996). Moreover, the NS1A protein of influenza A virus, otherFeat=[]-->, belongsTo=parr
61	specifically inhibits export of cellular but not viral mRNAs, otherFeat=[]-->, belongsTo=parr
62	by targeting two essential components of the pre-mRNA 3, otherFeat=[]-->, belongsTo=parr
63	end processing machinery (Nemeroff et al., 1998). Although, otherFeat=[]-->, belongsTo=parr
64	three yeast polyadenylation factors, Hrp1/Nab4, Nab2, and, otherFeat=[]-->, belongsTo=parr
65	Pab1 (Kessler et al., 1997; Hector et al., 2002; Brune et al.,, otherFeat=[]-->, belongsTo=parr
66	2005), and the mammalian nuclear poly(A) tail? binding pro-, otherFeat=[]-->, belongsTo=parr
67	tein (PABPN1, Calado et al., 2000), are nucleocytoplasmic, otherFeat=[]-->, belongsTo=parr
68	shuttling proteins, to date there is no evidence for a direct, otherFeat=[]-->, belongsTo=parr
69	role of 3 end processing factors in mRNA export., otherFeat=[]-->, belongsTo=parr
70	The mature 3 ends of most eukaryotic mRNAs are gen-, otherFeat=[]-->, belongsTo=parr
71	erated by endonucleolytic cleavage of the primary transcript, otherFeat=[]-->, belongsTo=parr
72	followed by the addition of a poly(A) tail to the upstream, otherFeat=[]-->, belongsTo=parr
73	cleavage product (for reviews see Zhao et al., 1999; Gilmartin,, otherFeat=[]-->, belongsTo=parr
74	2005). Cleavage Factor Im (CF Im) is a component of the 3, otherFeat=[]-->, belongsTo=parr
75	end processing complex that participates in the cleavage, otherFeat=[]-->, belongsTo=parr
76	reaction. CFIm is a heterodimer composed of a small subunit, otherFeat=[]-->, belongsTo=parr
77	of 25 kDa and a large subunit of 59, 68, or 72 kDa (Ru?eg-, otherFeat=[]-->, belongsTo=parr
78	segger et al., 1998). The 25- and 68-kDa subunits have been, otherFeat=[]-->, belongsTo=parr
79	This article was published online ahead of print in MBC in Press, otherFeat=[]-->, belongsTo=parrnote
80	(http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E09 ? 05? 0389), otherFeat=[]-->, belongsTo=parrnote
81	on October 28, 2009., otherFeat=[]-->, belongsTo=parrnote
82	These authors contributed equally to this work., otherFeat=[]-->, belongsTo=parrnote
83	Present addresses: ?Lawrence Berkeley National Laboratory, 1 Cy-, otherFeat=[]-->, belongsTo=parrnote
84	clotron Road, Mailstop Stanley 922, Berkeley, CA 94720; Medical, otherFeat=[]-->, belongsTo=parrnote
85	Research Council Human Genetics Unit, Western General Hospital,, otherFeat=[]-->, belongsTo=parrnote
86	Edinburgh EH4 2XU, United Kingdom., otherFeat=[]-->, belongsTo=parrnote
87	Address correspondence to: Silvia M.L. Barabino: (silvia.barabino@, otherFeat=[]-->, belongsTo=parrnote
88	unimib.it)., otherFeat=[]-->, belongsTo=parrnote
89	? 2009 by The American Society for Cell Biology, otherFeat=[]-->, belongsTo=nota_cab_pie
90	5211, otherFeat=[]-->, belongsTo=nota_cab_pie
91	shown to be sufficient to reconstitute CFIm activity for, otherFeat=[]-->, belongsTo=parr
92	poly(A) site cleavage in vitro upon addition to partially, otherFeat=[]-->, belongsTo=parr
93	purified 3 processing factors (Ru?egsegger et al., 1998). Both, otherFeat=[]-->, belongsTo=parr
94	subunits contact the RNA substrate, as demonstrated by UV, otherFeat=[]-->, belongsTo=parr
95	cross-linking studies (Ru?gsegger et al., 1996). The structure, otherFeat=[]-->, belongsTo=parr
96	of the CFIm 68-kDa subunit is strikingly similar to that of the, otherFeat=[]-->, belongsTo=parr
97	SR family of proteins, which plays an essential role in basal, otherFeat=[]-->, belongsTo=parr
98	and regulated pre-mRNA splicing (reviewed by Graveley,, otherFeat=[]-->, belongsTo=parr
99	2000). The 68-kDa protein possesses an N-terminal RNP-, otherFeat=[]-->, belongsTo=parr
100	type RNA recognition motif (RRM) and an RS-like C-termi-, otherFeat=[]-->, belongsTo=parr
101	nal region enriched in RS/D/E dipeptides that is reminis-, otherFeat=[]-->, belongsTo=parr
102	cent of the RS domain of SR proteins. The RS-like domain of, otherFeat=[]-->, belongsTo=parr
103	CF Im 68-kDa subunit is sufficient for the localization in, otherFeat=[]-->, belongsTo=parr
104	nuclear speckles (and in the nucleoplasm) and mediates the, otherFeat=[]-->, belongsTo=parr
105	interaction in vitro with a subset of shuttling SR proteins, otherFeat=[]-->, belongsTo=parr
106	(Dettwiler et al., 2004; Cardinale et al., 2007)., otherFeat=[]-->, belongsTo=parr
107	Here, we demonstrate that CF Im68 is a nucleocytoplasmic, otherFeat=[]-->, belongsTo=parr
108	shuttling protein that can stimulate mRNA export. First, we, otherFeat=[]-->, belongsTo=parr
109	show that CF Im68 is associated with components of the EJC., otherFeat=[]-->, belongsTo=parr
110	Second, we establish that CF Im68 interacts with the mRNA, otherFeat=[]-->, belongsTo=parr
111	export factor NXF1. Finally, we demonstrate that CF Im68 is, otherFeat=[]-->, belongsTo=parr
112	directly involved in mRNA export. Although CF Im68 can, otherFeat=[]-->, belongsTo=parr
113	stimulate export when tethered to a luciferase reporter, otherFeat=[]-->, belongsTo=parr
114	mRNA and its overexpression results in an increase of en-, otherFeat=[]-->, belongsTo=parr
115	dogenous mRNAs in the cytoplasmic fraction, RNAi-medi-, otherFeat=[]-->, belongsTo=parr
116	ated silencing leads to the retention of polyadenylated tran-, otherFeat=[]-->, belongsTo=parr
117	scripts in the nucleus. Furthermore, we show by sucrose, otherFeat=[]-->, belongsTo=parr
118	gradient centrifugation that CF Im68 cosediments with the, otherFeat=[]-->, belongsTo=parr
119	80S ribosome particle. CF Im68 is a well-established compo-, otherFeat=[]-->, belongsTo=parr
120	nent of the pre-mRNA 3 end processing complex. Our data, otherFeat=[]-->, belongsTo=parr
121	showing that CF Im68 is part of the mature mRNP particles, otherFeat=[]-->, belongsTo=parr
122	and contributes to their export to the cytoplasm highlight a, otherFeat=[]-->, belongsTo=parr
123	novel function for CF Im68 and provide a link between, otherFeat=[]-->, belongsTo=parr
124	pre-mRNA 3 end formation and mRNA export., otherFeat=[]-->, belongsTo=parr
125	MATERIALS AND METHODS, otherFeat=['U']-->, belongsTo=parr
126	Oligonucleotides, Plasmids, and Antibodies, otherFeat=[]-->, belongsTo=parr
127	Sequences for real-time RT-PCR probes and description of plasmids and, otherFeat=[]-->, belongsTo=parrnote
128	antibodies are presented in Supplemental Material., otherFeat=[]-->, belongsTo=parrnote
129	Cell Culture and Heterokaryon Assay, otherFeat=[]-->, belongsTo=parr
130	HeLa and HEK293 cells were cultured in DMEM supplemented with 10% FBS, otherFeat=[]-->, belongsTo=parrnote
131	and transfected using Effectene Transfection Reagent (Qiagen, Chatsworth,, otherFeat=[]-->, belongsTo=parrnote
132	CA) or Escort V (Sigma-Aldrich, St. Louis, MO) according to the manufac-, otherFeat=[]-->, belongsTo=parrnote
133	turer's instructions. Drug treatments were carried out as follows: actinomycin, otherFeat=[]-->, belongsTo=parrnote
134	D treatment (5 g/ml, Sigma-Aldrich) for2hor leptomycin B (LMB; 25, otherFeat=[]-->, belongsTo=parrnote
135	g/ml, Sigma-Aldrich) for1hin DMEM, followed by fixation and fluores-, otherFeat=[]-->, belongsTo=parrnote
136	cence microscopy., otherFeat=[]-->, belongsTo=parrnote
137	For heterokaryon nucleocytoplasmic shuttling assays, HeLa cells were tran-, otherFeat=[]-->, belongsTo=parrnote
138	siently transfected with different vectors as described. Twenty-four hours, otherFeat=[]-->, belongsTo=parrnote
139	after transfection, an equal number of HeLa cells was seeded onto the same, otherFeat=[]-->, belongsTo=parrnote
140	coverslip as NIH3T3 cells. The cells were incubated for3hinthe presence of, otherFeat=[]-->, belongsTo=parrnote
141	50 g/ml cycloheximide and for 30 min in the presence of 100 g/ml, otherFeat=[]-->, belongsTo=parrnote
142	cycloheximide before fusion. Cell fusions were done as described (Pinol-, otherFeat=[]-->, belongsTo=parrnote
143	Roma and Dreyfuss, 1992). Heterokaryons were incubated further for2hin, otherFeat=[]-->, belongsTo=parrnote
144	media containing 100 g/ml cycloheximide before fixation., otherFeat=[]-->, belongsTo=parrnote
145	Immunoprecipitations, otherFeat=[]-->, belongsTo=parr
146	After transfection, HEK293 cells were washed and harvested. For the immu-, otherFeat=[]-->, belongsTo=parrnote
147	noprecipitations of Flag-hUpf3b complexes, 8 100-mm dishes of transfected, otherFeat=[]-->, belongsTo=parrnote
148	cells were used for each assay. The pellets were quickly frozen in liquid, otherFeat=[]-->, belongsTo=parrnote
149	nitrogen, thawed, and resuspended in lysis buffer (50 mM Tris HCl, pH 8, 150, otherFeat=[]-->, belongsTo=parrnote
150	mM NaCl, 1% NP40, and 1 protease inhibitor cocktail [PIC]; Roche, India-, otherFeat=[]-->, belongsTo=parrnote
151	napolis, IN) and then incubated on ice for 1 h with occasional shaking. For the, otherFeat=[]-->, belongsTo=parrnote
152	coimmunoprecipitation of hemagglutinin (HA)-CF Im68 with either Flag-CF, otherFeat=[]-->, belongsTo=parrnote
153	Im25 or Flag-NXF1, cells were lysed in high-salt lysis buffer (50 mM Tris-HCl,, otherFeat=[]-->, belongsTo=parrnote
154	pH 8.0, 250 mM NaCl, 1% NP40). The samples were then centrifuged at 15,000, otherFeat=[]-->, belongsTo=parrnote
155	rcf for 15 min at 4?C, and the supernatants were collected. RNase A digestion, otherFeat=[]-->, belongsTo=parrnote
156	(200 mg/ml, Roche) was performed for 30 min at 30?C., otherFeat=[]-->, belongsTo=parrnote
157	Flag-tagged proteins were immunoprecipitated from the precleared, RNase, otherFeat=[]-->, belongsTo=parrnote
158	A?treated cell lysate with M2 anti-Flag agarose (Sigma-Aldrich) at 4?C for 2 h, otherFeat=[]-->, belongsTo=parrnote
159	on a rotator wheel in IP150 buffer (10 mM MgCl2, 10% NP40, 150 mM NaCl)., otherFeat=[]-->, belongsTo=parrnote
160	Then samples were centrifuged ten times at 600 g for 10 min at 4?C and, otherFeat=[]-->, belongsTo=parrnote
161	washed five times with 500 l of IP150 or IP250 buffer. Precipitated proteins, otherFeat=[]-->, belongsTo=parrnote
162	were eluted either with SDS sample buffer or M2 peptide and analyzed by, otherFeat=[]-->, belongsTo=parrnote
163	Western blotting. Detection was performed with an ECL detection kit (Am-, otherFeat=[]-->, belongsTo=parrnote
164	ersham, Piscataway, NJ)., otherFeat=[]-->, belongsTo=parrnote
165	GST-Fusion Protein Purification and GST-Pulldown, otherFeat=[]-->, belongsTo=parr
166	Assays, otherFeat=[]-->, belongsTo=parr
167	To study protein?protein interactions in vitro, GST fusion proteins were, otherFeat=[]-->, belongsTo=parrnote
168	expressed in Escherichia coli BL21(DE3)LysS or BL21(DE3) RIPL transformed, otherFeat=[]-->, belongsTo=parrnote
169	with pGEX-derived plasmids encoding glutathione S-transferase (GST; neg-, otherFeat=[]-->, belongsTo=parrnote
170	ative control) or GST-fusions with CFIm25, NXF1, or NXF1?202. After puri-, otherFeat=[]-->, belongsTo=parrnote
171	fication over glutathione Sepharose 4B beads (GE Healthcare, Waukesha, WI),, otherFeat=[]-->, belongsTo=parrnote
172	GST-CFIm25 and GST-NXF1 were further purified by gel filtration over a, otherFeat=[]-->, belongsTo=parrnote
173	Superdex 75 column (GE Healthcare)., otherFeat=[]-->, belongsTo=parrnote
174	GST pulldown assays with hexahistidine-tagged CF Im68 were performed, otherFeat=[]-->, belongsTo=parrnote
175	as follows. The purified GST fusions were coupled to glutathione Sepharose, otherFeat=[]-->, belongsTo=parrnote
176	4B beads, by using equimolar amounts of either GST-CF Im25 or GST-NXF1, otherFeat=[]-->, belongsTo=parrnote
177	(1.5 and 3 g, respectively) and 3 g of GST as negative control. The beads, otherFeat=[]-->, belongsTo=parrnote
178	and proteins were incubated in phosphate-buffered saline (PBS; 137 mM, otherFeat=[]-->, belongsTo=parrnote
179	NaCl, 2.7 mM KCl, 10 mM Na2HPO4,2mMKH2PO4) supplemented with, otherFeat=[]-->, belongsTo=parrnote
180	0.1% NP40 (PBS/NP40) at 4?C for 1.5 h with gentle agitation on a wheel,, otherFeat=[]-->, belongsTo=parrnote
181	followed by the addition of 400 ng histidine-tagged CF Im68 and incubation, otherFeat=[]-->, belongsTo=parrnote
182	for 1.5 h at 4?C. Subsequently, the beads were washed with PBS/NP40, and, otherFeat=[]-->, belongsTo=parrnote
183	the bound material was analyzed by SDS-PAGE and subsequent Western, otherFeat=[]-->, belongsTo=parrnote
184	blotting. Because input one-fifth of the added recombinant CF Im68 was, otherFeat=[]-->, belongsTo=parrnote
185	loaded on the gel, His-CF Im68 was detected by incubation of the blot with a, otherFeat=[]-->, belongsTo=parrnote
186	mouse monoclonal anti-His antibody (HIS1, Sigma-Aldrich) and a species-, otherFeat=[]-->, belongsTo=parrnote
187	specific horseradish peroxidase? coupled antibody (Promega, Madison, WI), otherFeat=[]-->, belongsTo=parrnote
188	and developed by the enhanced chemiluminescence method (ECL-Plus, GE, otherFeat=[]-->, belongsTo=parrnote
189	Healthcare). To visualize the bound recombinant GST-fusions, the membrane, otherFeat=[]-->, belongsTo=parrnote
190	was stained with Coomassie Brilliant Blue R-250., otherFeat=[]-->, belongsTo=parrnote
191	GST pulldown assays with in vitro?translated proteins were performed as, otherFeat=[]-->, belongsTo=parrnote
192	follows. The purified GST-fusions were coupled to glutathione Sepharose 4B, otherFeat=[]-->, belongsTo=parrnote
193	beads (GE Healthcare) in PBS/NP40 for 1 h and subsequently incubated with, otherFeat=[]-->, belongsTo=parrnote
194	RNAse treated [35S]methionine-labeled proteins obtained by coupled in vitro, otherFeat=[]-->, belongsTo=parrnote
195	transcription/translation in rabbit reticulocyte lysate (TNT T7 kit, Promega)., otherFeat=[]-->, belongsTo=parrnote
196	For nonradioactive in vitro translation, methionine was added to a final, otherFeat=[]-->, belongsTo=parrnote
197	concentration of 20 ?30 M. The samples were incubated in PBS supple-, otherFeat=[]-->, belongsTo=parrnote
198	mented with 0.1% NP40 at 4?C for 2 h with gentle agitation. Subsequently, otherFeat=[]-->, belongsTo=parrnote
199	beads were washed with PBS/NP40 (NP40 concentration increased to 1%),, otherFeat=[]-->, belongsTo=parrnote
200	and input and the bound fraction were analyzed by SDS-PAGE and detected, otherFeat=[]-->, belongsTo=parrnote
201	on a storm 820 Phosphorimager (Amersham)., otherFeat=[]-->, belongsTo=parrnote
202	Tethered mRNA Export Assay, otherFeat=[]-->, belongsTo=parr
203	Assays were carried out as described in Hargous et al. (2006) and Tintaru et al., otherFeat=[]-->, belongsTo=parrnote
204	(2007), with the exception that renilla luciferase rather than ?-galactosidase, otherFeat=[]-->, belongsTo=parrnote
205	was used for the normalization of transfection efficiency. For each trans-, otherFeat=[]-->, belongsTo=parrnote
206	fection, 700 ng of each of the plasmids encoding the MS2-protein, 50 ng, otherFeat=[]-->, belongsTo=parrnote
207	of luc-RRE firefly construct, and 5 ng of pRL-TK, a thymidine kinase renilla, otherFeat=[]-->, belongsTo=parrnote
208	luciferase control vector, were cotransfected in 24-well plates. Detection of, otherFeat=[]-->, belongsTo=parrnote
209	luciferase activity was performed with the Dual-luciferase Reporter Assay, otherFeat=[]-->, belongsTo=parrnote
210	System (Promega) according to the manufacturer's instruction. Luminescence, otherFeat=[]-->, belongsTo=parrnote
211	measurements were performed by using a Berthold luminometer. Four inde-, otherFeat=[]-->, belongsTo=parrnote
212	pendent sets of transfections were carried out in triplicate with two different, otherFeat=[]-->, belongsTo=parrnote
213	plasmids preparations. The average normalized luciferase activity in all the, otherFeat=[]-->, belongsTo=parrnote
214	experiments was calculated and expressed as percentage of the activity mea-, otherFeat=[]-->, belongsTo=parrnote
215	sured for REF., otherFeat=[]-->, belongsTo=parrnote
216	For analysis of the tethering experiments on the RNA-level, 1.4 106 HeLa, otherFeat=[]-->, belongsTo=parrnote
217	cells were transfected with 10 g MS2 fusion plasmid and 500 ng of pLuc-, otherFeat=[]-->, belongsTo=parrnote
218	SalRRE-6MS2 using Dreamfect (OZ Biosciences, Marseilles, France). The cells, otherFeat=[]-->, belongsTo=parrnote
219	were harvested 48 h after transfection. Nuclei were isolated as described, otherFeat=[]-->, belongsTo=parrnote
220	below and RNA was prepared by using an "Absolutely RNA RT-PCR Mini-, otherFeat=[]-->, belongsTo=parrnote
221	prep Kit" (Stratagene, La Jolla, CA). RNA, 1 g, was reverse-transcribed with, otherFeat=[]-->, belongsTo=parrnote
222	random hexamers and StrataScript 6.0 reverse transcriptase (Stratagene) ac-, otherFeat=[]-->, belongsTo=parrnote
223	cording to the manufacturer's protocol. Real-time RT-PCR was performed as, otherFeat=[]-->, belongsTo=parrnote
224	described below., otherFeat=[]-->, belongsTo=parrnote
225	Fluorescent In Situ Hybridization, otherFeat=[]-->, belongsTo=parr
226	For the visualization of the luciferase reporter RNA, the fluorescent in situ, otherFeat=[]-->, belongsTo=parrnote
227	hybridization (FISH) probes were 390 nt biotinylated antisense RNA mole-, otherFeat=[]-->, belongsTo=parrnote
228	cules transcribed in vitro from pRRE-Luc linearized with EcoRV with the, otherFeat=[]-->, belongsTo=parrnote
229	BioArray HighYield RNA Transcript Labeling Kit (Enzo Life Sciences, New, otherFeat=[]-->, belongsTo=parrnote
230	York, NY). HeLa cells were transiently transfected with pLUCRRE6MS2, otherFeat=[]-->, belongsTo=parrnote
231	reporter alone or cotransfected with pCNMS2CFIm68GFP, pCNMS2GFP,, otherFeat=[]-->, belongsTo=parrnote
232	pCNMS2TAP, pCNMS2REF, or pCNMS2REF-RRM. After 30 h, the cells were, otherFeat=[]-->, belongsTo=parrnote
233	fixed and FISH was performed according to standard protocols. Briefly, cells, otherFeat=[]-->, belongsTo=parrnote
234	were incubated in prehybridization buffer (2 SSC, 20% formamide, 0.2%, otherFeat=[]-->, belongsTo=parrnote
235	BSA, 1 g/ l tRNA) for 30 min at 37?C and then in hybridization solution (2, otherFeat=[]-->, belongsTo=parrnote
236	SSC, 20% formamide, 0.2% BSA, 10% dextran sulfate, 1 g/ l tRNA) in the, otherFeat=[]-->, belongsTo=parrnote
237	M.-D. Ruepp et al., otherFeat=[]-->, belongsTo=nota_cab_pie
238	Molecular Biology of the Cell, otherFeat=[]-->, belongsTo=nota_cab_pie
239	5212, otherFeat=[]-->, belongsTo=nota_cab_pie
240	presence of the biotinylated RNA probe (50 ng/slide) for3hat 37?C. Strin-, otherFeat=[]-->, belongsTo=parrnote
241	gent washes were performed in order to wash out unlabeled probe (twice, otherFeat=[]-->, belongsTo=parrnote
242	with 2 SSC 20% formamide, twice with 2 SSC, once with 1 SSC for 15, otherFeat=[]-->, belongsTo=parrnote
243	min at 45?C, once with 0.5 SSC for 15 min at 45?C, once with 0.5 SSC, otherFeat=[]-->, belongsTo=parrnote
244	0.3% NP40 for 2 min at 72?C, once with 2 SSC 0.1% NP40 for 1 min at, otherFeat=[]-->, belongsTo=parrnote
245	room temperature (RT), and once with 2 SSC for 10 min at RT). After a, otherFeat=[]-->, belongsTo=parrnote
246	preincubation wash with 4 SSC 0.1% Triton X-100 for 5 min at RT, the, otherFeat=[]-->, belongsTo=parrnote
247	FISH probe was revealed with 3 g/ml streptavidin-APC conjugate (BD, otherFeat=[]-->, belongsTo=parrnote
248	Biosciences, San Diego, CA) diluted in 4 SSC, 1% BSA for1hatRT. Finally,, otherFeat=[]-->, belongsTo=parrnote
249	cells were stained with DAPI and mounted with FluorSave reagent (Calbio-, otherFeat=[]-->, belongsTo=parrnote
250	chem, La Jolla, CA). Images were collected with a TCS SP2 AOBS confocal, otherFeat=[]-->, belongsTo=parrnote
251	microscope (Leica Microsystems, Exton, PA) and by using LSC software., otherFeat=[]-->, belongsTo=parrnote
252	Twelve-bit images were acquired by using the same setting parameters for all, otherFeat=[]-->, belongsTo=parrnote
253	the samples (gain, offset); for each field, five different xy sections along the, otherFeat=[]-->, belongsTo=parrnote
254	z-axis were acquired. FISH quantification was carried out with the same LCS, otherFeat=[]-->, belongsTo=parrnote
255	software. Measurements of the FISH probe were obtained for the nuclear, otherFeat=[]-->, belongsTo=parrnote
256	fluorescence (Sn), the total cell fluorescence (Sc), the area of the nucleus (An),, otherFeat=[]-->, belongsTo=parrnote
257	and area of the cell (Ac). The cytoplasmic (C ) amount of mRNA was calcu-, otherFeat=[]-->, belongsTo=parrnote
258	lated as follows: C 1 [(An Sn)/(Ac Sc)]. C/N ratios were calculated as, otherFeat=[]-->, belongsTo=parrnote
259	C/N C /(1 C ) (for reference see Valencia et al., 2008)., otherFeat=[]-->, belongsTo=parrnote
260	For the visualization of poly(A) RNA, HeLa cells were grown in six-well, otherFeat=[]-->, belongsTo=parrnote
261	plates on coverslips and transfected with the different plasmids, as indicated., otherFeat=[]-->, belongsTo=parrnote
262	Seven days past transfection, the cells were washed two times in PBS (137 mM, otherFeat=[]-->, belongsTo=parrnote
263	NaCl, 2.7 mM KCl, 10 mM Na2HPO4,2mMKH2PO4) before they were fixed, otherFeat=[]-->, belongsTo=parrnote
264	for 10 min at RT in 3.7% paraformaldehyde (PFA). After a wash in PBS for 5, otherFeat=[]-->, belongsTo=parrnote
265	min, the cells were permeabilized for 10 min with 100% methanol, followed, otherFeat=[]-->, belongsTo=parrnote
266	by a 10-min incubation in 70% ethanol, and a 5-min incubation in 1 M Tris, pH, otherFeat=[]-->, belongsTo=parrnote
267	8.0. The cells were then blocked for 30 min at 40?C in prewarmed prehybrid-, otherFeat=[]-->, belongsTo=parrnote
268	ization solution (2 x SSC, 20% formamide, 0.2% BSA, 1 g/ l tRNA in, otherFeat=[]-->, belongsTo=parrnote
269	DEPC-H2O). Prewarmed hybridization solution (2 SSC, 20% formamide,, otherFeat=[]-->, belongsTo=parrnote
270	10% dextran sulfate, 0.2% BSA, 1 g/ l tRNA, 1 ng/ l Cy3-labeled oligo-, otherFeat=[]-->, belongsTo=parrnote
271	(dT)50 in DEPC-H2O) was added to the coverslips and incubated at 37?C for, otherFeat=[]-->, belongsTo=parrnote
272	2 h. Next, the cells were washed twice with prewarmed 2 SSC and 20%, otherFeat=[]-->, belongsTo=parrnote
273	formamide in PBS, twice with prewarmed 2 SSC in PBS for 5 min each at, otherFeat=[]-->, belongsTo=parrnote
274	42?C and once in 1 SSC in PBS for 5 min at RT. Before the coverslips were, otherFeat=[]-->, belongsTo=parrnote
275	mounted (Mowiol 4 ? 88, Calbiochem, containing 0.25 g DAPI/ml), they, otherFeat=[]-->, belongsTo=parrnote
276	were washed three times for 5 min in PBS., otherFeat=[]-->, belongsTo=parrnote
277	RNA Interference, otherFeat=[]-->, belongsTo=parr
278	HeLa cells stably transfected with pcDNA3-?-globin cDNA were transfected, otherFeat=[]-->, belongsTo=parrnote
279	either with two siRNAs against CF Im68 or NXF1 (Silencer Select Pre-De-, otherFeat=[]-->, belongsTo=parrnote
280	signed & Validated siRNA, Applied Biosystems, Foster City, CA) at 25 nM, otherFeat=[]-->, belongsTo=parrnote
281	concentration each or a nontargeting siRNA control at 50 nM concentration,, otherFeat=[]-->, belongsTo=parrnote
282	by using Lullaby reagent (OZ Biosciences) according to the manufacturer's, otherFeat=[]-->, belongsTo=parrnote
283	instructions. After splitting, cells were subjected to a second round of trans-, otherFeat=[]-->, belongsTo=parrnote
284	fection with 50% of the amount of siRNAs indicated above. Cells depleted, otherFeat=[]-->, belongsTo=parrnote
285	from CF Im68 were harvested 4 d after the first transfection for analysis,, otherFeat=[]-->, belongsTo=parrnote
286	whereas cells depleted from NXF1 were harvested 48 h after transfection., otherFeat=[]-->, belongsTo=parrnote
287	Real-Time PCR Analysis, otherFeat=[]-->, belongsTo=parr
288	For the analysis of the nuclear and cytoplasmic distribution of mRNAs, HeLa, otherFeat=[]-->, belongsTo=parrnote
289	cells were cotransfected with pSUPuro constructs or pcDNA3-HA? derived, otherFeat=[]-->, belongsTo=parrnote
290	plasmids containing a puromycin selection marker by using Dreamfect (Oz, otherFeat=[]-->, belongsTo=parrnote
291	Biosciences). Culturing the cells in the presence of 1.5 g/ml puromycin, otherFeat=[]-->, belongsTo=parrnote
292	eliminated untransfected cells. Three days after transfection cells were, otherFeat=[]-->, belongsTo=parrnote
293	harvested., otherFeat=[]-->, belongsTo=parrnote
294	Nuclear-cytoplasmic fractionations of HeLa cells were performed as de-, otherFeat=[]-->, belongsTo=parrnote
295	scribed elsewhere (Carneiro and Schibler, 1984), except that RNase A treat-, otherFeat=[]-->, belongsTo=parrnote
296	ment was omitted. From each fraction, 20 l aliquots were taken for Western, otherFeat=[]-->, belongsTo=parrnote
297	blot analysis. Subsequently RNA was isolated by using TRI-Reagent (Am-, otherFeat=[]-->, belongsTo=parrnote
298	bion, Applied Biosystems, Inc., Foster City, CA), and DNA contamination, otherFeat=[]-->, belongsTo=parrnote
299	was removed by using Turbo DNA-free (Ambion). Total RNA, 2 g, from, otherFeat=[]-->, belongsTo=parrnote
300	each fraction was reverse transcribed with Stratascript reverse transcriptase, otherFeat=[]-->, belongsTo=parrnote
301	(Stratagene) according to the manufacturer's instruction. cDNA correspond-, otherFeat=[]-->, belongsTo=parrnote
302	ing to 40 ng RNA was amplified with specific primers and TaqMan probes in, otherFeat=[]-->, belongsTo=parrnote
303	an ABI SDS7000 Sequence Detection System (Applied Biosystems). The rela-, otherFeat=[]-->, belongsTo=parrnote
304	tive mRNA levels of the cytoplasmic and nuclear fractions were normalized, otherFeat=[]-->, belongsTo=parrnote
305	to 18S rRNA levels. The RT-PCR for X-ist and MIC 2a was performed in 50 l, otherFeat=[]-->, belongsTo=parrnote
306	1 Fast Start Master Mix (Roche) supplemented with 400 nM f.c. of each, otherFeat=[]-->, belongsTo=parrnote
307	primer pair, and 38 cycles were performed., otherFeat=[]-->, belongsTo=parrnote
308	Polysome Profile, otherFeat=[]-->, belongsTo=parr
309	Sucrose gradient fractionation was performed as described in Sanford et al., otherFeat=[]-->, belongsTo=parrnote
310	(2004). Briefly, HEK293 cells were cotransfected with plasmids expressing, otherFeat=[]-->, belongsTo=parrnote
311	HA-CF Im68 and T7-ASF. After 24 h cells were washed and collected by short, otherFeat=[]-->, belongsTo=parrnote
312	centrifugation at 4,000 rpm at 4?C and resuspended cold lysis buffer (100 mM, otherFeat=[]-->, belongsTo=parrnote
313	NaCl, 10 mM MgCl2, 30 mM Tris HCl, pH 7.5, 1 mM DTT, 0.05% Triton X-100), otherFeat=[]-->, belongsTo=parrnote
314	containing 100 g/ml cycloheximide, RNase, and protease inhibitors. After 5, otherFeat=[]-->, belongsTo=parrnote
315	min on ice, the extract was centrifuged for 4 min at 12,000 g at 4?C to pellet, otherFeat=[]-->, belongsTo=parrnote
316	nuclei and debris. For negative controls, extracts were treated before sucrose, otherFeat=[]-->, belongsTo=parrnote
317	gradient analysis either with 30 mM EDTA for 10 min at 4?C on a rotator, otherFeat=[]-->, belongsTo=parrnote
318	wheel, followed by the addition of 2 g RNase A (Roche) and further, otherFeat=[]-->, belongsTo=parrnote
319	incubation for 30 min at 30?C, or with 20 mM puromycin for1hat4?C on a, otherFeat=[]-->, belongsTo=parrnote
320	rotator wheel. Supernatants were resolved on 15?50% sucrose gradients pre-, otherFeat=[]-->, belongsTo=parrnote
321	pared in (100 mM NaCl, 10 mM MgCl2, 10 mM Tris HCl, pH 7.5). The gradient, otherFeat=[]-->, belongsTo=parrnote
322	was centrifuged at 4?C at 38,000 rpm in a Beckman SW41 rotor for 3 h. After, otherFeat=[]-->, belongsTo=parrnote
323	centrifugation, fractions were collected from the bottom, precipitated with, otherFeat=[]-->, belongsTo=parrnote
324	TCA, and analyzed by immunoblotting., otherFeat=[]-->, belongsTo=parrnote
325	Online Supplemental Materials are as follows: Supplemental File 1: legends to, otherFeat=[]-->, belongsTo=parrnote
326	Supplemental Figures S1?S8; Supplemental File 2: Supplemental methods; Sup-, otherFeat=[]-->, belongsTo=parrnote
327	plemental Figure S1: CF Im is imported into the nucleus as a heterodimer;, otherFeat=[]-->, belongsTo=parrnote
328	Supplemental Figure S2: Nucleocytoplasmic shuttling is an active process;, otherFeat=[]-->, belongsTo=parrnote
329	Supplemental Figure S3: Western blot analysis of HEK293 cell extracts used in, otherFeat=[]-->, belongsTo=parrnote
330	the mRNA export assay; Supplemental Figure S4: Western blot assessing the, otherFeat=[]-->, belongsTo=parrnote
331	purity of the nuclear fractions used in the experiments shown in Figure 6D, otherFeat=[]-->, belongsTo=parrnote
332	and 7E; Supplemental Figure S5: NXF1 depletion increases nuclear levels of, otherFeat=[]-->, belongsTo=parrnote
333	?-globin mRNA; Supplemental Figure S6: sucrose gradient fractionation of, otherFeat=[]-->, belongsTo=parrnote
334	puromycin-treated extracts; Supplemental Figure S7: color version of Figure, otherFeat=[]-->, belongsTo=parrnote
335	1; and Supplemental Figure S8: color version of Figure 2., otherFeat=[]-->, belongsTo=parrnote
336	RESULTS, otherFeat=[]-->, belongsTo=parr
337	CF I m Large Subunits Are Nucleocytoplasmic Shuttling, otherFeat=[]-->, belongsTo=parr
338	Proteins, otherFeat=[]-->, belongsTo=parr
339	We reported previously that the 68-kDa subunit of CF Im, otherFeat=[]-->, belongsTo=parr
340	interacts with a subset of SR proteins that were shown to, otherFeat=[]-->, belongsTo=parr
341	shuttle continuously between the nucleus and the cytoplasm, otherFeat=[]-->, belongsTo=parr
342	(Dettwiler et al., 2004). In addition, the 25-kDa subunit in-, otherFeat=[]-->, belongsTo=parr
343	teracts with PABPN1, which is a nucleocytoplasmic shut-, otherFeat=[]-->, belongsTo=parr
344	tling protein (Calado et al., 2000). On the basis of these, otherFeat=[]-->, belongsTo=parr
345	observations we wanted to test whether also the CF Im, otherFeat=[]-->, belongsTo=parr
346	subunits shuttle between the nucleus and the cytoplasm. We, otherFeat=[]-->, belongsTo=parr
347	analyzed their migration in an interspecies heterokaryon, otherFeat=[]-->, belongsTo=parr
348	fusion assay. HeLa cells expressing HA-tagged CF Im68, otherFeat=[]-->, belongsTo=parr
349	were fused to mouse NIH3T3 cells in the presence of cyclo-, otherFeat=[]-->, belongsTo=parr
350	heximide to produce heterokaryons. Shuttling of HA-CF, otherFeat=[]-->, belongsTo=parr
351	Im68 would lead to its equilibration into the nuclei of fused, otherFeat=[]-->, belongsTo=parr
352	NIH3T3 cells. As a control, HeLa cells were cotransfected, otherFeat=[]-->, belongsTo=parr
353	with a plasmid expressing GFP-hnRNP A1, a well-known, otherFeat=[]-->, belongsTo=parr
354	shuttling protein, or GFP-hnRNP C, a protein that is always, otherFeat=[]-->, belongsTo=parr
355	restricted to the nucleus (Pinol-Roma et al., 1988). In a rep-, otherFeat=[]-->, belongsTo=parr
356	resentative heterokaryon, 2 h after fusion, HA-CF Im68 and, otherFeat=[]-->, belongsTo=parr
357	GFP-hnRNP A1 were present both in the two HeLa cell, otherFeat=[]-->, belongsTo=parr
358	nuclei and in the mouse cell nucleus (Figure 1A, top row). In, otherFeat=[]-->, belongsTo=parr
359	contrast, GFP? hnRNP C was restricted, as expected, to the, otherFeat=[]-->, belongsTo=parr
360	HeLa cell nucleus (Figure 1A, bottom row). Quantitative, otherFeat=[]-->, belongsTo=parr
361	analysis showed that NIH3T3 nuclei were positive for, otherFeat=[]-->, belongsTo=parr
362	HA-CF Im68 in all of the 51 heterokaryons examined. Thus,, otherFeat=[]-->, belongsTo=parr
363	although CF Im68 is nuclear at steady state, it is continuously, otherFeat=[]-->, belongsTo=parr
364	traversing the nuclear envelope. A similar assay was per-, otherFeat=[]-->, belongsTo=parr
365	formed with HeLa cells expressing either GFP-CF Im59 or, otherFeat=[]-->, belongsTo=parr
366	GFP-CF Im25. As shown in Figure 1B, although CF Im59 can, otherFeat=[]-->, belongsTo=parr
367	efficiently migrate into the mouse nucleus (Figure 1B, top, otherFeat=[]-->, belongsTo=parr
368	row), shuttling of CF Im25 is less efficient (Figure 1B, bottom, otherFeat=[]-->, belongsTo=parr
369	row). A possible explanation for the observed, inefficient, otherFeat=[]-->, belongsTo=parr
370	migration of CF Im25 into the mouse nucleus could be that, otherFeat=[]-->, belongsTo=parr
371	this subunit is not able to shuttle on its own and may be, otherFeat=[]-->, belongsTo=parr
372	imported into the nucleus only in association with one of the, otherFeat=[]-->, belongsTo=parr
373	endogenous larger polypeptides. Therefore, if GFP-CF Im25, otherFeat=[]-->, belongsTo=parr
374	is overexpressed but the endogenous large subunits are, otherFeat=[]-->, belongsTo=parr
375	limiting, this may result in poor nuclear import of GFP-CF, otherFeat=[]-->, belongsTo=parr
376	Im25. Cotransfection experiments with CFP-CF Im68 and, otherFeat=[]-->, belongsTo=parr
377	YFP-CF Im25 constructs in which both ORFs are expressed under, otherFeat=[]-->, belongsTo=parr
378	the same strong promoter (so that the two CF Im subunits should, otherFeat=[]-->, belongsTo=parr
379	be present in the cell in almost equimolar amounts and could, otherFeat=[]-->, belongsTo=parr
380	therefore efficiently dimerize) demonstrated that, under, otherFeat=[]-->, belongsTo=parr
381	these conditions, the CF Im 25-kDa subunit is imported, otherFeat=[]-->, belongsTo=parr
382	into nucleus more efficiently, in agreement with the ex-, otherFeat=[]-->, belongsTo=parr
383	planation proposed above (Supplemental Figure S1)., otherFeat=[]-->, belongsTo=parr
384	To identify the region involved in the export of CF Im68,, otherFeat=[]-->, belongsTo=parr
385	we analyzed the shuttling behavior of various domain de-, otherFeat=[]-->, belongsTo=parr
386	letion mutants fused to GFP (68 N, 68 RS, 68RS, and, otherFeat=[]-->, belongsTo=parr
387	Nucleocytoplasmic Shuttling of CF Im, otherFeat=[]-->, belongsTo=nota_cab_pie
388	Vol. 20, December 15, 2009, otherFeat=[]-->, belongsTo=nota_cab_pie
389	5213, otherFeat=[]-->, belongsTo=nota_cab_pie
390	68RRM/RS, depicted in Supplemental Figure S2; Dettwiler, otherFeat=[]-->, belongsTo=parr
391	et al., 2004) at 37?C or, in addition, at 4?C to check for passive, otherFeat=[]-->, belongsTo=parr
392	diffusion. The rationale for this assay is that, at 4?C, both, otherFeat=[]-->, belongsTo=parr
393	receptor-mediated nuclear import and export are blocked,, otherFeat=[]-->, belongsTo=parr
394	whereas passive diffusion continues to occur. Temperature-, otherFeat=[]-->, belongsTo=parr
395	shift experiments were performed as described in Michael et, otherFeat=[]-->, belongsTo=parr
396	al. (1995). As shown in Supplemental Figure S2, all the, otherFeat=[]-->, belongsTo=parr
397	mutants were able to shuttle at 37?C but were restricted to, otherFeat=[]-->, belongsTo=parr
398	the HeLa cell nucleus at 4?C., otherFeat=[]-->, belongsTo=parr
399	Nuclear Export of CF Im68 is CRM1-independent and, otherFeat=[]-->, belongsTo=parr
400	Requires Active Transcription, otherFeat=[]-->, belongsTo=parr
401	The most common mechanism for the nuclear export of, otherFeat=[]-->, belongsTo=parr
402	proteins in eukaryotic cells is based on CRM1-dependent, otherFeat=[]-->, belongsTo=parr
403	systems. CRM1-mediated export is specifically blocked in, otherFeat=[]-->, belongsTo=parr
404	the presence of the fungal metabolite LMB, which inhibits, otherFeat=[]-->, belongsTo=parr
405	the formation of the ternary complex between CRM1, Ran-, otherFeat=[]-->, belongsTo=parr
406	GTP, and the cargo protein. To check whether CRM1 is, otherFeat=[]-->, belongsTo=parr
407	responsible for CF Im68 nuclear export, heterokaryon assays, otherFeat=[]-->, belongsTo=parr
408	were thus performed the presence of LMB. HeLa cells, otherFeat=[]-->, belongsTo=parr
409	treated in this way were transfected with plasmids express-, otherFeat=[]-->, belongsTo=parr
410	ing HA-CF Im68 in combination with GFP-HMGB1, a pro-, otherFeat=[]-->, belongsTo=parr
411	tein that requires CRM1 for export (Figure 2A). Treatment, otherFeat=[]-->, belongsTo=parr
412	with LMB specifically restricted HMGB1 to the HeLa cell, otherFeat=[]-->, belongsTo=parr
413	nucleus. while CF Im68 could still be efficiently exported, otherFeat=[]-->, belongsTo=parr
414	thus indicating that its export does not require CRM1., otherFeat=[]-->, belongsTo=parr
415	Previous studies have shown that CF Im is an RNA-binding, otherFeat=[]-->, belongsTo=parr
416	factor (Ru?egsegger et al., 1996). CF Im68 contacts the RNA, otherFeat=[]-->, belongsTo=parr
417	mainly via the charged C-terminal domain, whereas the RRM, otherFeat=[]-->, belongsTo=parr
418	is primarily involved in protein interactions with the small, otherFeat=[]-->, belongsTo=parr
419	subunit (Dettwiler et al., 2004). Therefore, it is possible that CF, otherFeat=[]-->, belongsTo=parr
420	Im68 is leaving the nucleus by "piggy-backing" on RNA mol-, otherFeat=[]-->, belongsTo=parr
421	ecules being exported by other factors, either in association, otherFeat=[]-->, belongsTo=parr
422	with the 25-kDa subunit or because of a direct interaction via, otherFeat=[]-->, belongsTo=parr
423	the C-terminal domain. To determine whether shuttling of CF, otherFeat=[]-->, belongsTo=parr
424	Im68 depends on mRNA synthesis, we performed hetero-, otherFeat=[]-->, belongsTo=parr
425	karyon assays in the presence of actinomycin D. As shown in, otherFeat=[]-->, belongsTo=parr
426	Figure 2B, GFP-CF Im68 shuttling is blocked in presence of the, otherFeat=[]-->, belongsTo=parr
427	transcription inhibitor, whereas relocalization of the protein in, otherFeat=[]-->, belongsTo=parr
428	cap-like structures around the nucleoli of the human cell can be, otherFeat=[]-->, belongsTo=parr
429	observed, as previously described (Cardinale et al., 2007; Figure, otherFeat=[]-->, belongsTo=parr
430	2B). Although we cannot formally rule out the possibility that, otherFeat=[]-->, belongsTo=parr
431	actinomycin D could affect the synthesis of a short-lived pro-, otherFeat=[]-->, belongsTo=parr
432	tein required for export, the most likely conclusion of these, otherFeat=[]-->, belongsTo=parr
433	experiments is that CF Im68 shuttling is dependent on mRNA, otherFeat=[]-->, belongsTo=parr
434	synthesis and possibly export. Therefore, on the basis of this, otherFeat=[]-->, belongsTo=parr
435	observation, we began to investigate a possible involvement of, otherFeat=[]-->, belongsTo=parr
436	CF Im68 in mRNA export., otherFeat=[]-->, belongsTo=parr
437	Figure 1. The two large CF Im subunits mi-, otherFeat=[]-->, belongsTo=fig_caption
438	grate between nuclei in interspecies hetero-, otherFeat=[]-->, belongsTo=fig_caption
439	karyons. (A) CF Im68 is a shuttling protein., otherFeat=[]-->, belongsTo=fig_caption
440	Left, merge of DAPI staining of HeLa and, otherFeat=[]-->, belongsTo=fig_caption
441	NIH3T3 nuclei (indicated by broken arrows), otherFeat=[]-->, belongsTo=fig_caption
442	and phase-contrast microscopy view. Middle,, otherFeat=[]-->, belongsTo=fig_caption
443	localization of GFP? hnRNP A1 or GFP-, otherFeat=[]-->, belongsTo=fig_caption
444	hnRNP C. Right, localization of HA-CF Im68., otherFeat=[]-->, belongsTo=fig_caption
445	(B) CF Im59 but not the 25-kDa subunit shut-, otherFeat=[]-->, belongsTo=fig_caption
446	tles between the nucleus and the cytoplasm., otherFeat=[]-->, belongsTo=fig_caption
447	Representative heterokaryons of HeLa cells,, otherFeat=[]-->, belongsTo=fig_caption
448	transfected with either GFP-CF Im59 or, otherFeat=[]-->, belongsTo=fig_caption
449	GFP-CF Im25. Broken arrows, the mouse nu-, otherFeat=[]-->, belongsTo=fig_caption
450	clei. A full-color version of this figure is avail-, otherFeat=[]-->, belongsTo=fig_caption
451	able as Supplementary Figure S7., otherFeat=[]-->, belongsTo=fig_caption
452	Figure 2. Export of CF Im68 depends on mRNA traffic. (A) Inhibition of the CRM1-mediated nuclear export pathway does not affect CF Im68, otherFeat=[]-->, belongsTo=fig_caption
453	shuttling activity. HeLa cells were cotransfected with expression constructs for HA-CF Im68 and GFP-HMGB1. Two hours before fusion with, otherFeat=[]-->, belongsTo=fig_caption
454	NHI3T3 cells and throughout the experiment, cells were incubated in the absence (not shown) or in the presence of LMB as described in, otherFeat=[]-->, belongsTo=fig_caption
455	Materials and Methods. HA-CF Im68 was detected by immunofluorescence. (B) HeLa cells transfected with GFP-CF Im68 and mouse NIH3T3, otherFeat=[]-->, belongsTo=fig_caption
456	cells were treated with actinomycin D and then fused as described in Materials and Methods. Left, merge of DAPI staining of HeLa and NIH3T3, otherFeat=[]-->, belongsTo=fig_caption
457	nuclei (indicated by broken arrows) and phase-contrast microscopy view. A full-color version of this figure is available as Supplementary, otherFeat=[]-->, belongsTo=fig_caption
458	Figure S8., otherFeat=[]-->, belongsTo=fig_caption
459	M.-D. Ruepp et al., otherFeat=[]-->, belongsTo=nota_cab_pie
460	Molecular Biology of the Cell, otherFeat=[]-->, belongsTo=nota_cab_pie
461	5214, otherFeat=[]-->, belongsTo=nota_cab_pie
462	CF I m68 Interacts with the mRNA Export Factor NXF1, otherFeat=[]-->, belongsTo=parr
463	The EJC, which is deposited on the mRNA by the splicing, otherFeat=[]-->, belongsTo=parr
464	reaction, consists of four core components (Y14, Magoh,, otherFeat=[]-->, belongsTo=parr
465	MLN51, and eIFAIII) and several more peripherally associ-, otherFeat=[]-->, belongsTo=parr
466	ated proteins (for review see Tange et al., 2004). Complexed, otherFeat=[]-->, belongsTo=parr
467	proteins include the splicing factors SRm160, RNPS1, Aci-, otherFeat=[]-->, belongsTo=parr
468	nus, SAP18 and Pinin, the mRNA export factors UAP56,, otherFeat=[]-->, belongsTo=parr
469	Aly/REF, and NXF1, and the NMD factor Upf3b. To deter-, otherFeat=[]-->, belongsTo=parr
470	mine if CF Im68 may be present in postsplicing complexes,, otherFeat=[]-->, belongsTo=parr
471	Upf3b-containing mRNPs were immunopurified from ex-, otherFeat=[]-->, belongsTo=parr
472	tracts of HEK293 cells transiently transfected with Flag?, otherFeat=[]-->, belongsTo=parr
473	tagged hUpf3b and analyzed by Western blotting with an-, otherFeat=[]-->, belongsTo=parr
474	tibodies against EJC proteins, hnRNP proteins, and with, otherFeat=[]-->, belongsTo=parr
475	anti-CF Im68 antiserum (Figure 3A). Because several of these, otherFeat=[]-->, belongsTo=parr
476	factors, including CF Im, can bind RNA, extracts were, otherFeat=[]-->, belongsTo=parr
477	treated with RNase A before immunoprecipitation to test for, otherFeat=[]-->, belongsTo=parr
478	true protein?protein interactions. RNase treatment was, otherFeat=[]-->, belongsTo=parr
479	shown to be effective by the disappearance of ?-actin mRNA, otherFeat=[]-->, belongsTo=parr
480	as revealed by RT-PCR (Figure 3A, left). Consistent with, otherFeat=[]-->, belongsTo=parr
481	previous reports, CBP80, NXF1, REF, and Magoh coimmu-, otherFeat=[]-->, belongsTo=parr
482	nopurified with Flag-hUpf3b (Figure 3A, right; Kim et al.,, otherFeat=[]-->, belongsTo=parr
483	2001; Lejeune et al., 2002; Singh et al., 2007). In addition to, otherFeat=[]-->, belongsTo=parr
484	these proteins, CF Im68 can also be detected in Upf3b-con-, otherFeat=[]-->, belongsTo=parr
485	taining mRNPs even in the presence of RNaseA, suggesting, otherFeat=[]-->, belongsTo=parr
486	that it must interact with at least one of the non-RNA com-, otherFeat=[]-->, belongsTo=parr
487	ponents of the complex. As expected, hnRNPA1, which is, otherFeat=[]-->, belongsTo=parr
488	an abundant component of heterogeneous nuclear RNP, otherFeat=[]-->, belongsTo=parr
489	(hnRNP) complexes but not of postsplicing complexes (Kim, otherFeat=[]-->, belongsTo=parr
490	et al., 2001), is not present in the pellet of the coimmunopre-, otherFeat=[]-->, belongsTo=parr
491	cipitation., otherFeat=[]-->, belongsTo=parr
492	Because export of CF Im68 is not mediated by CRM1 but, otherFeat=[]-->, belongsTo=parr
493	instead requires mRNA transcription, we asked whether, otherFeat=[]-->, belongsTo=parr
494	NXF1 might be the export receptor for CF Im68. Control, otherFeat=[]-->, belongsTo=parr
495	Flag-tagged CF Im25, -NXF1, -Upf1, -Upf3b, or the empty, otherFeat=[]-->, belongsTo=parr
496	Flag vector alone were transiently coexpressed with HA-, otherFeat=[]-->, belongsTo=parr
497	tagged CF Im68 in HEK293 cells. Interaction with CF Im68, otherFeat=[]-->, belongsTo=parrnote
498	was analyzed by immunoprecipitation of the cell lysates, otherFeat=[]-->, belongsTo=parr
499	with anti-Flag antibodies in the presence of RNase A fol-, otherFeat=[]-->, belongsTo=parr
500	lowed by Western blotting with anti-CF Im68 antiserum. The, otherFeat=[]-->, belongsTo=parr
501	expression level of Flag-NXF1 was significantly lower than, otherFeat=[]-->, belongsTo=parr
502	that of the other Flag-tagged proteins, and especially of, otherFeat=[]-->, belongsTo=parr
503	Flag-Upf1 (Figure 3B, input, cf. lanes 4 and 5). Nevertheless,, otherFeat=[]-->, belongsTo=parr
504	the amount of CF Im68 associated with Flag-NXF1 was far, otherFeat=[]-->, belongsTo=parr
505	greater than that obtained with either Flag-Upf3b or Flag, otherFeat=[]-->, belongsTo=parr
506	alone, and equivalent to the amount coprecipitated with, otherFeat=[]-->, belongsTo=parr
507	Flag-Upf1 (pellet, cf. lanes 8 ?11). These results suggest that, otherFeat=[]-->, belongsTo=parr
508	CF Im68 interacts specifically with NXF1 in vivo, and possi-, otherFeat=[]-->, belongsTo=parr
509	bly also with Upf1. Thus, to determine if the interaction, otherFeat=[]-->, belongsTo=parr
510	between CF Im68 and NXF1 is a direct one, we performed, otherFeat=[]-->, belongsTo=parr
511	GST pulldown experiments with purified recombinant pro-, otherFeat=[]-->, belongsTo=parr
512	teins. E. coli? expressed GST, control GST-CF Im25, or GST-, otherFeat=[]-->, belongsTo=parr
513	NXF1 were incubated with purified baculovirus-expressed,, otherFeat=[]-->, belongsTo=parr
514	histidine-tagged CF Im68. Bound proteins were analyzed by, otherFeat=[]-->, belongsTo=parr
515	Western blotting using anti-histidine (Figure 3C). Because, otherFeat=[]-->, belongsTo=parr
516	GST?NXF1 coprecipitated equivalent amount of CF Im68 to, otherFeat=[]-->, belongsTo=parr
517	that coprecipitated by CF Im25 (cf. lanes 2 and 3), we con-, otherFeat=[]-->, belongsTo=parr
518	clude that CF Im68 interacts specifically with the mRNA, otherFeat=[]-->, belongsTo=parr
519	export factor NXF1., otherFeat=[]-->, belongsTo=parr
520	We next aimed to identify the region in CF Im68 respon-, otherFeat=[]-->, belongsTo=parr
521	sible for the interaction with NXF1. We tested the interaction, otherFeat=[]-->, belongsTo=parr
522	Figure 3. CF Im68 and NXF1 interact in HEK293, otherFeat=[]-->, belongsTo=fig_caption
523	cells and in vitro. (A) CF Im68 associates with, otherFeat=[]-->, belongsTo=fig_caption
524	hUpf3b-containing protein complexes. Left, RT-, otherFeat=[]-->, belongsTo=fig_caption
525	PCR quantitation of the level of ?-actin mRNA be-, otherFeat=[]-->, belongsTo=fig_caption
526	fore or after RNase treatment. Extracts were treated, otherFeat=[]-->, belongsTo=fig_caption
527	with increasing amounts of RNaseA. Lane 1, un-, otherFeat=[]-->, belongsTo=fig_caption
528	treated extract; lane 2, 1 g/ml; lane 3, 3 g/ml;, otherFeat=[]-->, belongsTo=fig_caption
529	lane 4, 15 g/ml. Right, coimmunoprecipitation ex-, otherFeat=[]-->, belongsTo=fig_caption
530	periment of RNase A?treated extracts of HEK293, otherFeat=[]-->, belongsTo=fig_caption
531	cells transfected with Flag-Upf3b by using anti-Flag, otherFeat=[]-->, belongsTo=fig_caption
532	antibodies (lanes 3 and 5). For mock transfection,, otherFeat=[]-->, belongsTo=fig_caption
533	the same amount of Flag-pCMVTag2 was used as, otherFeat=[]-->, belongsTo=fig_caption
534	negative control (lanes 2 and 4). Coimmunoprecipi-, otherFeat=[]-->, belongsTo=fig_caption
535	tated proteins were visualized by Western blotting, otherFeat=[]-->, belongsTo=fig_caption
536	with the indicated antibodies. Lane 1, total extract, otherFeat=[]-->, belongsTo=fig_caption
537	from untransfected cells. (B) CF Im68 interacts with NXF1 in HEK293 cell extract. Coimmunoprecipitations with anti-Flag antibody of RNase, otherFeat=[]-->, belongsTo=fig_caption
538	A?treated extracts of HEK293 cells transfected with Flag-tagged CF Im25 (positive control, lanes 3 and 8), NXF1 (lanes 4 and 9), hUpf1 (lanes, otherFeat=[]-->, belongsTo=fig_caption
539	5 and 10), or hUpf3b (lanes 6 and 11), and HA-tagged CF Im68, as indicated. For mock transfections (lanes 2 and 7), the same amount of, otherFeat=[]-->, belongsTo=fig_caption
540	Flag-pCMVTag2 was used. Bound proteins (Pellet) were detected by Western blotting with anti-CF Im68 antiserum (top panel). To control, otherFeat=[]-->, belongsTo=fig_caption
541	for equal loading, the membrane was sequentially probed without stripping with anti-Flag and anti-actin antibodies (bottom panel). An, otherFeat=[]-->, belongsTo=fig_caption
542	asterisk indicates residual signal of the anti-CF Im68 antibody. Lane 1, total extract from untransfected cells. (C) Recombinant CF Im68, otherFeat=[]-->, belongsTo=fig_caption
543	interacts with NXF1 in vitro. Left, Coomassie-stained SDS-PAGE of the purified recombinant proteins used in the pulldown assay. Right, GST, otherFeat=[]-->, belongsTo=fig_caption
544	(lane 2), GST-tagged CF Im25 (lane 3) and NXF1 (lane 4) were tested for interaction with histidine-tagged CF Im68. Eluted proteins were, otherFeat=[]-->, belongsTo=fig_caption
545	analyzed by Western blotting with anti-histidine antibody., otherFeat=[]-->, belongsTo=fig_caption
546	Nucleocytoplasmic Shuttling of CF Im, otherFeat=[]-->, belongsTo=nota_cab_pie
547	Vol. 20, December 15, 2009, otherFeat=[]-->, belongsTo=nota_cab_pie
548	5215, otherFeat=[]-->, belongsTo=nota_cab_pie
549	by coimmunoprecipitation of Flag-tagged NXF1 with GFP, otherFeat=[]-->, belongsTo=parr
550	fusions of wild-type and mutant CF Im68 that were coex-, otherFeat=[]-->, belongsTo=parr
551	pressed in HEK293 cells (Figure 4B). Tested mutants in-, otherFeat=[]-->, belongsTo=parr
552	cluded a deletion of the N-terminal portion of CFIm68, a, otherFeat=[]-->, belongsTo=parr
553	region that contains the RRM and was shown to bind the, otherFeat=[]-->, belongsTo=parr
554	25-kDa subunit (68 N), a deletion of the entire RS-like re-, otherFeat=[]-->, belongsTo=parr
555	gion at the C-terminus (68 RS), the RS-like domain alone, otherFeat=[]-->, belongsTo=parr
556	(68RS), and a fusion of the RRM and the RS domains, otherFeat=[]-->, belongsTo=parr
557	(68RRM/RS). As shown in Figure 4B Flag-NXF1 precipi-, otherFeat=[]-->, belongsTo=parr
558	tated the fragments 68RRM/RS and 68 RS. Deletion of the, otherFeat=[]-->, belongsTo=parr
559	N-terminal portion impaired the interaction with NXF1,, otherFeat=[]-->, belongsTo=parr
560	whereas the RS-like domain appeared to interact with NXF1, otherFeat=[]-->, belongsTo=parr
561	albeit very inefficiently. Similar results were obtained in GST, otherFeat=[]-->, belongsTo=parr
562	pulldown experiments in which recombinant GST-CF Im68, otherFeat=[]-->, belongsTo=parr
563	truncations were incubated with Flag-NXF1 expressed in, otherFeat=[]-->, belongsTo=parr
564	HEK293 cells (data not shown)., otherFeat=[]-->, belongsTo=parr
565	To confirm the requirement of the N-terminal region of CF, otherFeat=[]-->, belongsTo=parr
566	Im68 for the direct interaction with NXF1, in vitro binding, otherFeat=[]-->, belongsTo=parr
567	assays were performed. Glutathione beads prebound with, otherFeat=[]-->, belongsTo=parr
568	GST or GST-NXF1 were incubated with 35S-labeled CF Im68, otherFeat=[]-->, belongsTo=parr
569	or 68 N. Bound fractions were resolved and visualized by, otherFeat=[]-->, belongsTo=parr
570	autoradiography. As shown in Figure 4C, only the full-, otherFeat=[]-->, belongsTo=parr
571	length protein, but not the N-terminally deleted one, bound, otherFeat=[]-->, belongsTo=parr
572	NXF1, indicating that the NXF1 interaction region lies, otherFeat=[]-->, belongsTo=parr
573	within the first 213 amino acids of CF Im68., otherFeat=[]-->, belongsTo=parr
574	NXF1 can be functionally divided into three domains, otherFeat=[]-->, belongsTo=parr
575	(Figure 5A; Izaurralde, 2002): the N-terminal half (aa 1-372), otherFeat=[]-->, belongsTo=parr
576	interacts with REF (Stutz et al., 2000); the region between aa, otherFeat=[]-->, belongsTo=parr
577	371 and 551 binds the essential export cofactor p15 (Katahira, otherFeat=[]-->, belongsTo=parr
578	et al., 1999; Guzik et al., 2001; Izaurralde, 2002); and the, otherFeat=[]-->, belongsTo=parr
579	C-terminal domain interacts with components of the NPC, otherFeat=[]-->, belongsTo=parr
580	(Bachi et al., 2000). Shuttling SR proteins interact with the, otherFeat=[]-->, belongsTo=parr
581	Figure 4. CF Im68 interacts with NXF1 via its N terminus. (A) Schematic representation, otherFeat=[]-->, belongsTo=fig_caption
582	of the domain structure of CF Im68. The open and solid boxes indicate the regions of the, otherFeat=[]-->, belongsTo=fig_caption
583	protein present in each mutant relative to the 552 amino acid wild-type protein shown, otherFeat=[]-->, belongsTo=fig_caption
584	at the top. The connecting line indicates missing residues (222? 419) in the RRM/RS, otherFeat=[]-->, belongsTo=fig_caption
585	protein. (B) HEK293 cells were transfected either with pCMVTag2-Flag or with pFlag-, otherFeat=[]-->, belongsTo=fig_caption
586	NXF1 and plasmids expressing GFP-tagged full-length CF Im68 (lanes 2? 4), or the, otherFeat=[]-->, belongsTo=fig_caption
587	domain deletion mutant proteins 68 N (lanes 5?7), 68RRM/RS (lanes 8 ?10), 68RS (lanes, otherFeat=[]-->, belongsTo=fig_caption
588	11?13), and 68 RS (lanes 14 ?16). Total extracts were immunoprecipitated with anti-Flag antibody and analyzed by Western blotting. Lane, otherFeat=[]-->, belongsTo=fig_caption
589	1, extract from untransfected cells. (C) E. coli? expressed GST or GST-NXF1 prebound to beads were individually incubated with in, otherFeat=[]-->, belongsTo=fig_caption
590	vitro?translated, 35S-labeled HA-tagged CF Im68 or 68 N. Bound fractions (lanes 2, 3, 5, and 6) were subjected to SDS-PAGE and analyzed, otherFeat=[]-->, belongsTo=fig_caption
591	by autoradiography., otherFeat=[]-->, belongsTo=fig_caption
592	Figure 5. CF Im68 and SR proteins do, otherFeat=[]-->, belongsTo=fig_caption
593	not bind the same region of NXF1. (A), otherFeat=[]-->, belongsTo=fig_caption
594	NXF1 domain (top) organization with the, otherFeat=[]-->, belongsTo=fig_caption
595	202?aa fragment (bottom). The regions in-, otherFeat=[]-->, belongsTo=fig_caption
596	teracting with the SR proteins, REF, p15,, otherFeat=[]-->, belongsTo=fig_caption
597	and the NPC are indicated. (B) SR pro-, otherFeat=[]-->, belongsTo=fig_caption
598	teins and CF Im68 do not bind to the same, otherFeat=[]-->, belongsTo=fig_caption
599	NXF1 region. Left, E. coli? expressed GST,, otherFeat=[]-->, belongsTo=fig_caption
600	GST-NXF1, or GST-NXF1 202 were incu-, otherFeat=[]-->, belongsTo=fig_caption
601	bated with in vitro?translated, 35S-labeled, otherFeat=[]-->, belongsTo=fig_caption
602	CF Im68. Bound fractions were analyzed, otherFeat=[]-->, belongsTo=fig_caption
603	by autoradiography. Left, Coomassie-, otherFeat=[]-->, belongsTo=fig_caption
604	stained gel of the purified recombinant proteins. (C) SR proteins can bridge the interaction between NXF1 and CF Im68. E. coli? expressed GST,, otherFeat=[]-->, belongsTo=fig_caption
605	or GST-NXF1 were incubated with in vitro?translated, 35S-labeled CF Im68 N in the presence of recombinant, histidine-tagged hTra2? as, otherFeat=[]-->, belongsTo=fig_caption
606	described in Materials and Methods. Bound fractions were analyzed by autoradiography., otherFeat=[]-->, belongsTo=fig_caption
607	M.-D. Ruepp et al., otherFeat=[]-->, belongsTo=nota_cab_pie
608	Molecular Biology of the Cell, otherFeat=[]-->, belongsTo=nota_cab_pie
609	5216, otherFeat=[]-->, belongsTo=nota_cab_pie
610	same domain of NXF1 that binds REF (Huang et al., 2003). To, otherFeat=[]-->, belongsTo=parr
611	determine if CF Im68 and SR proteins bind the same domain, otherFeat=[]-->, belongsTo=parr
612	of NXF1, glutathione beads loaded with GST or GST-NXF1, otherFeat=[]-->, belongsTo=parr
613	were individually incubated with in vitro?translated, 35S-, otherFeat=[]-->, belongsTo=parrnote
614	labeled, HA-tagged CF Im68 or 68 N. As shown in Figure 5B, otherFeat=[]-->, belongsTo=parr
615	only full-length NXF1, but not the first 202 aa, which bind, otherFeat=[]-->, belongsTo=parr
616	shuttling SR proteins (Huang et al., 2003 and data not, otherFeat=[]-->, belongsTo=parr
617	shown), interacts with CF Im68., otherFeat=[]-->, belongsTo=parr
618	We previously demonstrated that the C-terminal charged, otherFeat=[]-->, belongsTo=parr
619	domain of CF I m68 interacts with shuttling SR proteins, otherFeat=[]-->, belongsTo=parr
620	(Dettwiler et al., 2004). Thus, we tested whether the, otherFeat=[]-->, belongsTo=parr
621	C-terminal region of CF Im68 may mediate the formation of, otherFeat=[]-->, belongsTo=parr
622	a ternary complex between SR proteins and NXF1. In the, otherFeat=[]-->, belongsTo=parr
623	presence of the SR protein hTra2?, GST-NXF1 can indeed, otherFeat=[]-->, belongsTo=parr
624	efficiently select 35S-labeled 68 N, which on its own did not, otherFeat=[]-->, belongsTo=parr
625	bind NXF1 (cf. Figures 4C and 5C)., otherFeat=[]-->, belongsTo=parr
626	CF I m68 Stimulates mRNA Export, otherFeat=[]-->, belongsTo=parr
627	To test if CF Im68 may contribute to general mRNA export, otherFeat=[]-->, belongsTo=parr
628	through the interaction with the receptor protein NXF1 (as is, otherFeat=[]-->, belongsTo=parr
629	the case for REF and some SR proteins), we used a recently, otherFeat=[]-->, belongsTo=parr
630	described, tethered mRNA export assay (Hargous et al.,, otherFeat=[]-->, belongsTo=parr
631	2006). In this assay, an mRNA export factor is expressed as, otherFeat=[]-->, belongsTo=parr
632	a fusion protein with a bacteriophage MS2 coat protein tag, otherFeat=[]-->, belongsTo=parr
633	and then artificially tethered to a reporter RNA containing, otherFeat=[]-->, belongsTo=parr
634	the luciferase ORF and six MS2 coat protein? binding sites, otherFeat=[]-->, belongsTo=parr
635	within an inefficiently spliced intron (Figure 6A). The bind-, otherFeat=[]-->, belongsTo=parr
636	ing of the export factor leads to the nuclear export of the, otherFeat=[]-->, belongsTo=parr
637	unspliced RNA that would otherwise be retained in the, otherFeat=[]-->, belongsTo=parr
638	nucleus, resulting in the expression of luciferase activity. We, otherFeat=[]-->, belongsTo=parr
639	therefore checked whether tethering CF Im68 protein in this, otherFeat=[]-->, belongsTo=parr
640	way would promote export of the reporter RNA. Vectors, otherFeat=[]-->, belongsTo=parr
641	expressing the different MS2 fusion proteins were trans-, otherFeat=[]-->, belongsTo=parr
642	fected into HEK293 cells together with the luciferase re-, otherFeat=[]-->, belongsTo=parr
643	porter construct. Cotransfection of a third vector encoding, otherFeat=[]-->, belongsTo=parr
644	renilla luciferase was used to control the transfection effi-, otherFeat=[]-->, belongsTo=parr
645	ciency. As controls, MS2 fusions of GFP, NXF1, REF, and, otherFeat=[]-->, belongsTo=parr
646	REF-RRM (aa 71?155; Hargous et al., 2006) were tested in the, otherFeat=[]-->, belongsTo=parr
647	same experiments. As seen in Figure 6B, direct tethering of, otherFeat=[]-->, belongsTo=parr
648	NXF1 resulted in a very strong luciferase activity. Impor-, otherFeat=[]-->, belongsTo=parr
649	tantly, tethering of CF Im68 led to a luciferase expression that, otherFeat=[]-->, belongsTo=parr
650	was 10 times lower than that elicited by NXF1, but still, otherFeat=[]-->, belongsTo=parr
651	higher than that of REF. As expected, REF-RRM did not, otherFeat=[]-->, belongsTo=parr
652	stimulate export of the RNA Control experiments revealed, otherFeat=[]-->, belongsTo=parr
653	Figure 6. Tethering of CFIm68 leads to mRNA export. (A) Schematic of, otherFeat=[]-->, belongsTo=fig_caption
654	pRRE-Luc construct. The position of the FISH probe is indicated. (B), otherFeat=[]-->, belongsTo=fig_caption
655	Normalized firefly/renilla luciferase activity generated by the MS2 fu-, otherFeat=[]-->, belongsTo=fig_caption
656	sion proteins in the tethered mRNA export assay. Error bars, SDs from, otherFeat=[]-->, belongsTo=fig_caption
657	four independent sets of assays. The averaged activity of each MS2-, otherFeat=[]-->, belongsTo=fig_caption
658	fusion protein was expressed as a percentage of the average activity, otherFeat=[]-->, belongsTo=fig_caption
659	measured for MS2-REF calculated as described in Materials and Methods., otherFeat=[]-->, belongsTo=fig_caption
660	RRE, transfection with pRRE-Luc without MS2 fusion protein. (C) FISH, otherFeat=[]-->, belongsTo=fig_caption
661	of HeLa cells cotransfected with pRRE-Luc and double-tagged GFP/, otherFeat=[]-->, belongsTo=fig_caption
662	MS2 (a?a ), GFP/MS2?CF Im68 (b? b ), or Myc/MS2-NXF1 (c and c )., otherFeat=[]-->, belongsTo=fig_caption
663	DAPI staining was used to identify the cell nucleus (a? c), localization of, otherFeat=[]-->, belongsTo=fig_caption
664	RRE-Luc mRNA in representative cells was visualized by FISH (a ?c ); GFP fluorescence was used to identify cells expressing the exogenous, otherFeat=[]-->, belongsTo=fig_caption
665	protein (a and b ). (D) The average cytoplasmic to nuclear (C/N) ratio for the RRE-Luc mRNA was determined by quantitative analysis of, otherFeat=[]-->, belongsTo=fig_caption
666	FISH experiments as shown in C for the indicated number of cells per construct. Error bars, SDs. (E) Real-time PCR analysis of the relative, otherFeat=[]-->, belongsTo=fig_caption
667	amount of RRE-Luc mRNA in the nuclear fractions of cells transfected with the indicated MS2-fusions constructs as determined by real-time, otherFeat=[]-->, belongsTo=fig_caption
668	RT-PCR. Three experiments were performed and average values are shown., otherFeat=[]-->, belongsTo=fig_caption
669	Nucleocytoplasmic Shuttling of CF Im, otherFeat=[]-->, belongsTo=nota_cab_pie
670	Vol. 20, December 15, 2009, otherFeat=[]-->, belongsTo=nota_cab_pie
671	5217, otherFeat=[]-->, belongsTo=nota_cab_pie
672	Figure 7. CF Im68 promotes export of mRNAs. (A) Hela cells growing on coverslips, overexpressing HA-tagged CFIm68 and a puromycin, otherFeat=[]-->, belongsTo=fig_caption
673	resistance as well as control cells expressing only the puromycin resistance were fixed with formaldehyde and permeabilized with methanol., otherFeat=[]-->, belongsTo=fig_caption
674	Polyadenylated mRNA was visualized by hybridization of a Cy3-labeled oligo(dT)50 probe, whereas the nuclei were stained with DAPI. The, otherFeat=[]-->, belongsTo=fig_caption
675	panels are projections of Z-stacks from top to bottom of the nuclei. Images were collected on a Leica TCS SP2 AOBS laser scanning confocal, otherFeat=[]-->, belongsTo=fig_caption
676	microscope equipped with a HCX PL APOlbd.BL 63.0 1.2W objective (Leica Microsystems). (B) PCR analysis of nuclear and cytoplasmic, otherFeat=[]-->, belongsTo=fig_caption
677	RNAs of the nuclear X-ist mRNA (top) and the X-encoded Mic 2A transcript (bottom). (C) Overxpression of CF Im68 stimulates mRNA export., otherFeat=[]-->, belongsTo=fig_caption
678	Real-time PCR analysis of the relative amount of ?-actin, Gapdh, Upf2, and Smg7 mRNA levels in the cytoplasmic (top histogram) and, otherFeat=[]-->, belongsTo=fig_caption
679	nuclear (bottom histogram) fraction of cells expressing HA-tagged EGFP ( ) or HA-tagged CFIm68 (u). The values were normalized to 18S, otherFeat=[]-->, belongsTo=fig_caption
680	rRNA levels and then divided by the values obtained in cells expressing the EGFP control. Three independent experiments were performed, otherFeat=[]-->, belongsTo=fig_caption
681	and average values of three real-time PCR runs with cDNAs from one representative experiment are shown. (D) HeLa cells stably expressing, otherFeat=[]-->, belongsTo=fig_caption
682	?-globin mRNA were transfected with a control siRNA or with siRNAs against CFIm68. Total extract was prepared and analyzed by Western, otherFeat=[]-->, belongsTo=fig_caption
683	blotting with anti-CFIm68 and with anti-SmB/B antibodies. Three different experiments are shown. (E) CFIm68 depletion increases nuclear, otherFeat=[]-->, belongsTo=fig_caption
684	levels of ?-globin mRNA. Real-time RT-PCR analysis of the relative amount of ?-globin mRNA in the nuclear fraction of HeLa cells, otherFeat=[]-->, belongsTo=fig_caption
685	transfected with a control siRNA or cells depleted of CFIm68. The indicated values are normalized to 18S rRNA levels. Three experiments, otherFeat=[]-->, belongsTo=fig_caption
686	were performed and average values are shown. (F) CFIm68 depletion does not affect pre-mRNA 3 end processing. Real-time RT-PCR of, otherFeat=[]-->, belongsTo=fig_caption
687	?-actin mRNA was performed with probes spanning the 5 UTR or the cleavage site to measure total and unprocessed mRNA, respectively., otherFeat=[]-->, belongsTo=fig_caption
688	The ratio of precursor to total mRNA (pre/tot) was calculated to assess the efficiency of 3 end processing. The processing activity of cells, otherFeat=[]-->, belongsTo=fig_caption
689	transfected with the control siRNA was set to 1., otherFeat=[]-->, belongsTo=fig_caption
690	M.-D. Ruepp et al., otherFeat=[]-->, belongsTo=nota_cab_pie
691	Molecular Biology of the Cell, otherFeat=[]-->, belongsTo=nota_cab_pie
692	5218, otherFeat=[]-->, belongsTo=nota_cab_pie
693	that all the MS2 fusion proteins were expressed (Western, otherFeat=[]-->, belongsTo=parr
694	blot; Supplemental Figure S3) and correctly localized to the, otherFeat=[]-->, belongsTo=parr
695	cell nucleus (immunofluorescence microscopy, data not, otherFeat=[]-->, belongsTo=parr
696	shown)., otherFeat=[]-->, belongsTo=parr
697	Mutant CF Im68 proteins were also tested by this assay., otherFeat=[]-->, belongsTo=parr
698	Deletion of the N-terminal domain (68 N) that is necessary, otherFeat=[]-->, belongsTo=parr
699	for the direct interaction with NXF1 in vitro did not signif-, otherFeat=[]-->, belongsTo=parr
700	icantly affect mRNA export, most likely because the protein, otherFeat=[]-->, belongsTo=parr
701	can still interact with NXF1 via SR protein(s) bound to the, otherFeat=[]-->, belongsTo=parr
702	C-terminus (see above). In contrast, the deletion of 2/3 of the, otherFeat=[]-->, belongsTo=parr
703	C-terminal RS motif (68 C) strongly reduced luciferase ex-, otherFeat=[]-->, belongsTo=parr
704	pression. However, because this fragment is known to be, otherFeat=[]-->, belongsTo=parr
705	imported into the nucleus inefficiently (Dettwiler et al., 2004),, otherFeat=[]-->, belongsTo=parr
706	this result must be interpreted with caution. In further con-, otherFeat=[]-->, belongsTo=parr
707	trol experiments, the 30- and the 73-kDa subunits of the, otherFeat=[]-->, belongsTo=parr
708	cleavage and polyadenylation specificity factor CPSF had no, otherFeat=[]-->, belongsTo=parr
709	effect when tethered to the reporter RNA (Figure 6B and, otherFeat=[]-->, belongsTo=parr
710	data not shown, respectively)., otherFeat=[]-->, belongsTo=parr
711	The assay described above relies on the measurement of, otherFeat=[]-->, belongsTo=parr
712	luciferase enzymatic activity and thus requires mRNA trans-, otherFeat=[]-->, belongsTo=parr
713	lation. To rule out that CF Im68 might affect the translation, otherFeat=[]-->, belongsTo=parr
714	efficiency rather than RNA export, we sought to measure, otherFeat=[]-->, belongsTo=parr
715	directly nuclear and cytoplasmic mRNA levels in the pres-, otherFeat=[]-->, belongsTo=parr
716	ence of varying amounts of CF Im68. First, steady-state, otherFeat=[]-->, belongsTo=parr
717	pRRE-Luc mRNA levels were visualized by FISH; Figure, otherFeat=[]-->, belongsTo=parr
718	6C). Quantitation of FISH images for individual cells re-, otherFeat=[]-->, belongsTo=parr
719	vealed that the cytoplasmic to nuclear (C/N) ratio of the, otherFeat=[]-->, belongsTo=parr
720	FISH signal was 1 for the GFP control, 2.28 for GFP-CF, otherFeat=[]-->, belongsTo=parr
721	Im68, and 3.54 for NXF1 (Figure 6D). Additionally, we car-, otherFeat=[]-->, belongsTo=parr
722	ried out quantitative reverse-transcription PCR (qPCR) anal-, otherFeat=[]-->, belongsTo=parr
723	ysis of the relative amount of luciferase mRNA in the nu-, otherFeat=[]-->, belongsTo=parr
724	clear fractions of cells transfected with different MS2-fusion, otherFeat=[]-->, belongsTo=parr
725	constructs (Figure 6E). Quantitation of the relative nuclear, otherFeat=[]-->, belongsTo=parr
726	luciferase RNA levels showed that tethering of MS2-CF Im68, otherFeat=[]-->, belongsTo=parr
727	decreased the amount of transcript in the nucleus by 2.5-, otherFeat=[]-->, belongsTo=parr
728	fold compared with MS2-REF, whereas the two CF Im68, otherFeat=[]-->, belongsTo=parr
729	deletion mutants (68 N and 68 C) were less effective in, otherFeat=[]-->, belongsTo=parr
730	promoting mRNA export. The nuclear fractions were devoid, otherFeat=[]-->, belongsTo=parr
731	of cytoplasmic contaminations as shown by Western blot, otherFeat=[]-->, belongsTo=parr
732	analyses for lamin A/C (nuclear) and tyrosine tubulin (cy-, otherFeat=[]-->, belongsTo=parr
733	toplasmic; Supplemental Figure S4). Taken together, these, otherFeat=[]-->, belongsTo=parr
734	experiments indicate that tethering of CF Im68 can promote, otherFeat=[]-->, belongsTo=parr
735	mRNA export., otherFeat=[]-->, belongsTo=parr
736	To further characterize this new function of CF Im68, we, otherFeat=[]-->, belongsTo=parr
737	investigated the effect of its overexpression on the export of, otherFeat=[]-->, belongsTo=parr
738	endogenous mRNAs. HeLa cells were transfected with a, otherFeat=[]-->, belongsTo=parr
739	selectable plasmid driving CF Im68 expression under the, otherFeat=[]-->, belongsTo=parr
740	CMV promoter, and the nucleo-cytoplasmic distribution of, otherFeat=[]-->, belongsTo=parr
741	polyadenylated RNAs was examined by FISH with a Cy3-, otherFeat=[]-->, belongsTo=parr
742	labeled oligo(dT) probe (Figure 7A). In cells overexpressing, otherFeat=[]-->, belongsTo=parr
743	CF Im68, a slight but appreciable reduction of the amount of, otherFeat=[]-->, belongsTo=parr
744	nuclear polyadenylated RNA could be observed. To analyze, otherFeat=[]-->, belongsTo=parr
745	the effect of CF Im68 overexpression on the nucleocytoplas-, otherFeat=[]-->, belongsTo=parr
746	mic transport of specific mRNAs, nuclear and cytoplasmic, otherFeat=[]-->, belongsTo=parr
747	fractions were prepared, and RNA was isolated from these, otherFeat=[]-->, belongsTo=parr
748	extracts. RT-PCR for the nuclear X-inactivation specific, otherFeat=[]-->, belongsTo=parr
749	(X-ist) mRNA revealed that no nuclear RNA had leaked into, otherFeat=[]-->, belongsTo=parr
750	the cytoplasmic fractions (Figure 7B). The amounts of four, otherFeat=[]-->, belongsTo=parr
751	different mRNAs were then measured by qPCR. They are, otherFeat=[]-->, belongsTo=parr
752	shown in Figure 7C as relative values normalized to the, otherFeat=[]-->, belongsTo=parr
753	amounts of the same transcripts measured in cells overex-, otherFeat=[]-->, belongsTo=parr
754	pressing HA-EGFP. Importantly, the nuclear concentration, otherFeat=[]-->, belongsTo=parr
755	of each of these transcripts was reduced two- to threefold,, otherFeat=[]-->, belongsTo=parr
756	whereas cytoplasmic levels were increased for three of them,, otherFeat=[]-->, belongsTo=parr
757	with the exception of ?-actin. Nevertheless, even for ?-actin, otherFeat=[]-->, belongsTo=parr
758	mRNA, the cytoplasmic to nuclear ratio increased 2.2-fold, otherFeat=[]-->, belongsTo=parr
759	compared with the control, indicating that CF Im68 overex-, otherFeat=[]-->, belongsTo=parr
760	pression stimulates mRNA export also in this case., otherFeat=[]-->, belongsTo=parr
761	We also determined the effect of CF Im68 depletion on, otherFeat=[]-->, belongsTo=parr
762	mRNA export. Silencing experiments were performed in, otherFeat=[]-->, belongsTo=parr
763	HeLa cells stably transfected with the ?-globin gene lacking, otherFeat=[]-->, belongsTo=parr
764	natural introns and therefore expressing cDNA transcripts, otherFeat=[]-->, belongsTo=parr
765	that do not undergo splicing (Valencia et al., 2008). The assay, otherFeat=[]-->, belongsTo=parr
766	was validated by depleting NXF1. Two days after transfec-, otherFeat=[]-->, belongsTo=parr
767	tion of specific siRNAs or of a control siRNA, mRNA levels, otherFeat=[]-->, belongsTo=parr
768	were analyzed by real-time RT-PCR. NXF1 mRNA was re-, otherFeat=[]-->, belongsTo=parr
769	duced by 92% compared with control siRNA-treated cells,, otherFeat=[]-->, belongsTo=parr
770	and, as a consequence, the level of the nuclear ?-globin, otherFeat=[]-->, belongsTo=parr
771	mRNA was increased 2.5 times (Supplemental Figure S5)., otherFeat=[]-->, belongsTo=parr
772	In the case of CF Im68, 4 d after transfection of two specific, otherFeat=[]-->, belongsTo=parr
773	siRNAs, CF Im68 mRNA was reduced by 93% compared, otherFeat=[]-->, belongsTo=parr
774	with control siRNA-treated cells, whereas the level of the, otherFeat=[]-->, belongsTo=parr
775	mRNA of the closely related 59-kDa subunit remained un-, otherFeat=[]-->, belongsTo=parr
776	changed (data not shown). Western blot analysis (Figure 7D), otherFeat=[]-->, belongsTo=parr
777	confirmed that the level of the remaining CF Im68 protein, otherFeat=[]-->, belongsTo=parr
778	was below detection. Importantly, this silencing of CF Im68, otherFeat=[]-->, belongsTo=parr
779	resulted in a small but statistically significant increase in the, otherFeat=[]-->, belongsTo=parr
780	nuclear level of ?-globin mRNA (Figure 7E). As above, the, otherFeat=[]-->, belongsTo=parr
781	purity of these nuclear fractions was assessed by Western, otherFeat=[]-->, belongsTo=parr
782	blot analyses for lamin A/C and tyrosine tubulin (Supple-, otherFeat=[]-->, belongsTo=parr
783	mental Figure S4). This result supports the idea that CF Im68, otherFeat=[]-->, belongsTo=parr
784	may act in mRNA export, similarly to SR proteins and/or, otherFeat=[]-->, belongsTo=parr
785	REF, by contributing to the recruitment of NXF1 to the, otherFeat=[]-->, belongsTo=parr
786	mRNA., otherFeat=[]-->, belongsTo=parr
787	Another variable that could potentially affect the nucleo-, otherFeat=[]-->, belongsTo=parr
788	cytoplasmic distribution of mRNAs is inefficient 3 end pro-, otherFeat=[]-->, belongsTo=parr
789	cessing that would lead to nuclear retention of the unproc-, otherFeat=[]-->, belongsTo=parr
790	essed transcript. However, CF Im68 depletion did not, otherFeat=[]-->, belongsTo=parr
791	significantly affect the 3 end processing efficiency of neither, otherFeat=[]-->, belongsTo=parr
792	endogenous ?-actin pre-mRNA (Figure 7F) nor of the ?-, otherFeat=[]-->, belongsTo=parr
793	globin cDNA transcript, which has the strong polyadenyla-, otherFeat=[]-->, belongsTo=parr
794	tion signal of bovine growth hormone (BGH, data not, otherFeat=[]-->, belongsTo=parr
795	shown)., otherFeat=[]-->, belongsTo=parr
796	In summary, the above experiments indicate a relevant, otherFeat=[]-->, belongsTo=parr
797	biological role for CF Im68 in the export of mRNAs., otherFeat=[]-->, belongsTo=parr
798	CF I m68 Cosediments with Ribosomal Particles, otherFeat=[]-->, belongsTo=parr
799	Some of the components of the EJC, including REF and, otherFeat=[]-->, belongsTo=parr
800	NXF1, dissociate from the mRNA upon entry into the cyto-, otherFeat=[]-->, belongsTo=parr
801	plasm (Le Hir et al., 2001). Therefore, it is possible that CF, otherFeat=[]-->, belongsTo=parr
802	Im68 is present in the mRNPs in the nucleus but may then, otherFeat=[]-->, belongsTo=parr
803	dissociate during or shortly after mRNA export. To gain, otherFeat=[]-->, belongsTo=parr
804	insight into the remodeling of CF Im68-containing mRNPs, otherFeat=[]-->, belongsTo=parr
805	after export, cytoplasmic fractions of HEK293 cells were, otherFeat=[]-->, belongsTo=parr
806	fractionated across 15?50% sucrose gradients, and the dis-, otherFeat=[]-->, belongsTo=parr
807	tribution of CF Im68 was determined by Western blotting., otherFeat=[]-->, belongsTo=parr
808	Figure 8A shows that most of the cytoplasmic CF Im68 was, otherFeat=[]-->, belongsTo=parr
809	found in lighter complexes at the top of the gradient. How-, otherFeat=[]-->, belongsTo=parr
810	ever, CF Im68 could also be detected in the 80S (monosome), otherFeat=[]-->, belongsTo=parr
811	region, although to a lesser extent than ASF/SF2. CF Im68, otherFeat=[]-->, belongsTo=parr
812	distribution correlated with that of the nuclear cap-binding, otherFeat=[]-->, belongsTo=parr
813	complex component CBP80 (Figure 8B). Treatment of cyto-, otherFeat=[]-->, belongsTo=parr
814	plasmic extracts with RNaseA, which induces dissociation of, otherFeat=[]-->, belongsTo=parr
815	mono- and polyribosomes into ribosomal subunits, led to a, otherFeat=[]-->, belongsTo=parr
816	redistribution of CF Im68, SF2/ASF, and rpS6 to the top of, otherFeat=[]-->, belongsTo=parr
817	the gradient (Figure 8C). Likewise, we found that cosedi-, otherFeat=[]-->, belongsTo=parr
818	mentation of CF Im68 with 80S particles was sensitive to, otherFeat=[]-->, belongsTo=parr
819	EDTA (data not shown). In contrast, treatment with puro-, otherFeat=[]-->, belongsTo=parr
820	mycin, which causes premature termination and thus the, otherFeat=[]-->, belongsTo=parr
821	disassembly of translating heavy polysomes and an increase, otherFeat=[]-->, belongsTo=parr
822	of the 80S peak (Sabatini et al., 1971) did not affect CF Im68, otherFeat=[]-->, belongsTo=parr
823	distribution (Supplemental Figure S6). We conclude that CF, otherFeat=[]-->, belongsTo=parr
824	Im68 is not associated with translating ribosomes but, like, otherFeat=[]-->, belongsTo=parr
825	Nucleocytoplasmic Shuttling of CF Im, otherFeat=[]-->, belongsTo=nota_cab_pie
826	Vol. 20, December 15, 2009, otherFeat=[]-->, belongsTo=nota_cab_pie
827	5219, otherFeat=[]-->, belongsTo=nota_cab_pie
828	CBP80 and PABP2, is part of the mRNP particle that is, otherFeat=[]-->, belongsTo=parr
829	remodeled in the cytoplasm during the initial stages of, otherFeat=[]-->, belongsTo=parr
830	translation when nuclear proteins are replaced by their cy-, otherFeat=[]-->, belongsTo=parr
831	toplasmic counterparts (Ishigaki et al., 2001; Dostie and, otherFeat=[]-->, belongsTo=parr
832	Dreyfuss, 2002)., otherFeat=[]-->, belongsTo=parr
833	DISCUSSION, otherFeat=[]-->, belongsTo=parr
834	Although stimulation of mRNA export by polyadenylation, otherFeat=[]-->, belongsTo=parr
835	has long been observed, so far little is known about the, otherFeat=[]-->, belongsTo=parr
836	possible mechanism. In this report we provide first evidence, otherFeat=[]-->, belongsTo=parr
837	that the 68-kDa subunit of CF Im, a pre-mRNA 3 end, otherFeat=[]-->, belongsTo=parr
838	processing factor, interacts with the mRNA export receptor, otherFeat=[]-->, belongsTo=parr
839	NXF1 and stimulates mRNA export. On the basis of our, otherFeat=[]-->, belongsTo=parr
840	observations and on previous data showing that CF Im68 is, otherFeat=[]-->, belongsTo=parr
841	associated with BrdU-labeled nascent transcripts (Cardinale, otherFeat=[]-->, belongsTo=parr
842	et al., 2007), we propose that CF Im68 is loaded onto the, otherFeat=[]-->, belongsTo=parr
843	pre-mRNA during cleavage and polyadenylation of the 3, otherFeat=[]-->, belongsTo=parr
844	end of the transcript and remains bound to the mRNA all the, otherFeat=[]-->, belongsTo=parr
845	way to the cytoplasm where it is removed by the translation, otherFeat=[]-->, belongsTo=parr
846	machinery. CF Im68 may thus act as a mark of correct 3 end, otherFeat=[]-->, belongsTo=parr
847	maturation and contribute to efficient mRNA export via, otherFeat=[]-->, belongsTo=parr
848	NXF1 recruitment., otherFeat=[]-->, belongsTo=parr
849	Role of RNA-binding Shuttling Proteins in mRNA Export, otherFeat=[]-->, belongsTo=parr
850	Several RNA binding proteins exhibit shuttling activity in-, otherFeat=[]-->, belongsTo=parr
851	cluding the splicing factor U2AF, the polyadenylation factor, otherFeat=[]-->, belongsTo=parr
852	PABPN1, and members of the hnRNP and SR protein fam-, otherFeat=[]-->, belongsTo=parr
853	ilies (for review see Gama-Carvalho and Carmo-Fonseca,, otherFeat=[]-->, belongsTo=parr
854	2001). Although U2AF, PABPN1, and the hnRNP proteins, otherFeat=[]-->, belongsTo=parr
855	Figure 8. CF Im68 cosediments with the translation machinery. (A), otherFeat=[]-->, belongsTo=fig_caption
856	HEK293 cell cytoplasmic extracts were fractionated across 15?50%, otherFeat=[]-->, belongsTo=fig_caption
857	sucrose gradients and analyzed by immunoblotting with ASF/SF2,, otherFeat=[]-->, belongsTo=fig_caption
858	CF Im68, and rpS6. (Top) UV absorbance (254 nm) profile of cyto-, otherFeat=[]-->, belongsTo=fig_caption
859	solic ribonucleoprotein complexes. (B) Fractions were additionally, otherFeat=[]-->, belongsTo=fig_caption
860	analyzed by immunoblotting with ASF/SF2, CF Im68, and in addi-, otherFeat=[]-->, belongsTo=fig_caption
861	tion CBP80 and PABPC1. (C) Sucrose gradient analysis of RNase, otherFeat=[]-->, belongsTo=fig_caption
862	A?treated cytoplasmic extracts. CF Im68, SF2/ASF, and rpS6 con-, otherFeat=[]-->, belongsTo=fig_caption
863	centrate very narrowly across the gradient of HEK293 cytoplasmic, otherFeat=[]-->, belongsTo=fig_caption
864	extracts treated with RNase A, which induces dissociation of ribo-, otherFeat=[]-->, belongsTo=fig_caption
865	somal subunits. (Top) UV absorbance (254 nm) profile of cytosolic, otherFeat=[]-->, belongsTo=fig_caption
866	ribonucleoprotein complexes., otherFeat=[]-->, belongsTo=fig_caption
867	M.-D. Ruepp et al., otherFeat=[]-->, belongsTo=nota_cab_pie
868	Molecular Biology of the Cell, otherFeat=[]-->, belongsTo=nota_cab_pie
869	5220, otherFeat=[]-->, belongsTo=nota_cab_pie
870	were shown to shuttle actively and independently of mRNA, otherFeat=[]-->, belongsTo=parr
871	traffic, shuttling SR proteins are exported bound to the, otherFeat=[]-->, belongsTo=parr
872	mRNA. Similar to SR proteins, CF Im shuttling activity de-, otherFeat=[]-->, belongsTo=parr
873	pends on active transcription, suggesting that it leaves the, otherFeat=[]-->, belongsTo=parr
874	nucleus in association with the mRNA. Consistent with this, otherFeat=[]-->, belongsTo=parr
875	observation both CF Im subunits contact the RNA substrate,, otherFeat=[]-->, belongsTo=parr
876	as demonstrated by UV cross-linking studies (Ru?egsegger et, otherFeat=[]-->, belongsTo=parr
877	al., 1996)., otherFeat=[]-->, belongsTo=parr
878	What is the role of shuttling proteins in mRNA export?, otherFeat=[]-->, belongsTo=parr
879	Shuttling RNA-binding proteins may contribute to the as-, otherFeat=[]-->, belongsTo=parr
880	sembly of an export competent mRNP by recruiting mRNA, otherFeat=[]-->, belongsTo=parr
881	export factors. Consistent with this view U2AF 35-kDa sub-, otherFeat=[]-->, belongsTo=parr
882	unit (Zolotukhin et al., 2002), several shuttling SR proteins, otherFeat=[]-->, belongsTo=parr
883	(Huang et al., 2003; Lai and Tarn, 2004), and now CF Im have, otherFeat=[]-->, belongsTo=parr
884	been shown to interact with the mRNA export receptor, otherFeat=[]-->, belongsTo=parr
885	NXF1. In addition, shuttling RNA-binding proteins may, otherFeat=[]-->, belongsTo=parr
886	fulfill different roles in the nucleus and in the cytoplasm., otherFeat=[]-->, belongsTo=parr
887	This is the case for instance for the shuttling SR protein, otherFeat=[]-->, belongsTo=parr
888	ASF/SF2 that, besides its well-characterized role in basal, otherFeat=[]-->, belongsTo=parr
889	and regulated splicing, is involved in mRNA surveillance, otherFeat=[]-->, belongsTo=parr
890	and in translation (Sanford et al., 2004; Zhang and Krainer,, otherFeat=[]-->, belongsTo=parr
891	2004)., otherFeat=[]-->, belongsTo=parr
892	Mechanisms for the Recruitment of the mRNA Export, otherFeat=[]-->, belongsTo=parr
893	Receptor NXF1, otherFeat=[]-->, belongsTo=parr
894	In S. cerevisiae where only 5% of the genes contain introns,, otherFeat=[]-->, belongsTo=parr
895	export factor recruitment is coupled to transcription and 3, otherFeat=[]-->, belongsTo=parr
896	end formation rather than to intron splicing. The interaction, otherFeat=[]-->, belongsTo=parr
897	between the mRNA and the heterodimeric export receptor, otherFeat=[]-->, belongsTo=parr
898	Mex67/Mtr2 (NXF1/p15 in metazoa) is provided by the, otherFeat=[]-->, belongsTo=parr
899	RNA-binding protein Yra1 (REF). Yra1 interacts with the, otherFeat=[]-->, belongsTo=parr
900	transcription elongation complex (THO complex, Strasser et, otherFeat=[]-->, belongsTo=parr
901	al., 2002; Abruzzi et al., 2004) and requires 3 end formation, otherFeat=[]-->, belongsTo=parr
902	to be recruited cotranscriptionally to mRNAs (Lei and Silver,, otherFeat=[]-->, belongsTo=parr
903	2002). How the yeast pre-mRNA 3 end processing complex, otherFeat=[]-->, belongsTo=parr
904	participates in the recruitment of mRNA export factors is,, otherFeat=[]-->, belongsTo=parr
905	however, still unknown., otherFeat=[]-->, belongsTo=parr
906	In metazoans, the current model for the recruitment of, otherFeat=[]-->, belongsTo=parr
907	export factors on the mRNA proposes that adaptor proteins, otherFeat=[]-->, belongsTo=parr
908	such as REF and the export receptor NXF1 are "deposited", otherFeat=[]-->, belongsTo=parr
909	after splicing as components of the EJC (for review see Stutz, otherFeat=[]-->, belongsTo=parr
910	and Izaurralde, 2003). REF is recruited by UAP56 that binds, otherFeat=[]-->, belongsTo=parr
911	cotranscriptionally. Before export, NXF1/p15 would bind to, otherFeat=[]-->, belongsTo=parr
912	REF and thereby release UAP56. However, only NXF1, p15,, otherFeat=[]-->, belongsTo=parr
913	and UAP56 have been shown so far to be essential for this, otherFeat=[]-->, belongsTo=parr
914	process (Tan et al., 2000; Herold et al., 2001; Wilkie et al., 2001;, otherFeat=[]-->, belongsTo=parr
915	Gatfield and Izaurralde, 2002; Wiegand et al., 2002). Re-, otherFeat=[]-->, belongsTo=parr
916	cently, an EJC-independent mechanism was proposed for, otherFeat=[]-->, belongsTo=parr
917	the recruitment of REF based on the observation that UAP56, otherFeat=[]-->, belongsTo=parr
918	and REF bind at the 5 end of the mRNA and interact with, otherFeat=[]-->, belongsTo=parr
919	the cap-binding complex. In this new model recruitment, otherFeat=[]-->, belongsTo=parr
920	occurs via an interaction between REF and the cap-binding, otherFeat=[]-->, belongsTo=parr
921	protein CBP80 (Cheng et al., 2006). The observation that, otherFeat=[]-->, belongsTo=parr
922	depletion of REF in Drosophila cells does not lead to, otherFeat=[]-->, belongsTo=parr
923	accumulation of poly(A)RNA in the nucleus (Gatfield and, otherFeat=[]-->, belongsTo=parr
924	Izaurralde, 2002) suggests that alternative mechanisms, otherFeat=[]-->, belongsTo=parr
925	must exist for the recruitment of NXF1 on mature mRNAs., otherFeat=[]-->, belongsTo=parr
926	Consistent with this idea, the shuttling SR proteins SRp20,, otherFeat=[]-->, belongsTo=parr
927	Figure 9. Model of potential role of CF Im68 in assembly and export of mRNPs. (a) CF Im is recruited to the pre-mRNA as part of the cleavage, otherFeat=[]-->, belongsTo=fig_caption
928	and polyadenylation complex. (b) Once correct 3 end processing has occurred, CF Im68 remains associated with the mRNA and (c) in, otherFeat=[]-->, belongsTo=fig_caption
929	conjunction with SR proteins contributes to the recruitment of the export receptor NXF1. (d) In the cytoplasm, the translating ribosomes, otherFeat=[]-->, belongsTo=fig_caption
930	remove the nuclear proteins that are reimported into the nucleus., otherFeat=[]-->, belongsTo=fig_caption
931	Nucleocytoplasmic Shuttling of CF Im, otherFeat=[]-->, belongsTo=nota_cab_pie
932	Vol. 20, December 15, 2009, otherFeat=[]-->, belongsTo=nota_cab_pie
933	5221, otherFeat=[]-->, belongsTo=nota_cab_pie
934	9G8, and ASF/SF2 that can bind to the pre-mRNA indepen-, otherFeat=[]-->, belongsTo=parr
935	dently of the EJC, were shown to act as NXF1-adaptor, otherFeat=[]-->, belongsTo=parr
936	proteins. Here, we have demonstrated that CF Im68 interacts, otherFeat=[]-->, belongsTo=parr
937	with NXF1 and contributes to mRNA export most likely in, otherFeat=[]-->, belongsTo=parr
938	conjunction with SR proteins. The presence of CF Im68 may, otherFeat=[]-->, belongsTo=parr
939	signal that the mRNA has been properly cleaved and poly-, otherFeat=[]-->, belongsTo=parr
940	adenylated (Figure 9) and may be packaged into an export-, otherFeat=[]-->, belongsTo=parr
941	competent mRNA ribonucleoprotein particle (mRNP) that, otherFeat=[]-->, belongsTo=parr
942	can recruit the export receptor. This novel observation pro-, otherFeat=[]-->, belongsTo=parr
943	vides for the first time a molecular connection between 3, otherFeat=[]-->, belongsTo=parr
944	end formation and mRNA export., otherFeat=[]-->, belongsTo=parr
945	In contrast to NXF1, CF Im68 does not appear to dissociate, otherFeat=[]-->, belongsTo=parr
946	from the mRNA upon entry into the cytoplasm. Instead,, otherFeat=[]-->, belongsTo=parr
947	sucrose gradient centrifugation shows that CF Im68 cosedi-, otherFeat=[]-->, belongsTo=parr
948	ments with the 80S ribosome. According to current models,, otherFeat=[]-->, belongsTo=parr
949	the first passage of the 80S ribosome on an mRNA (called, otherFeat=[]-->, belongsTo=parr
950	"pioneer round" of translation) leads to a major rearrange-, otherFeat=[]-->, belongsTo=parr
951	ment of the mRNP composition whereby the remaining, otherFeat=[]-->, belongsTo=parr
952	nuclear mRNP components are removed and replaced by, otherFeat=[]-->, belongsTo=parr
953	cytoplasmic factors (for review see Maquat, 2004; Moore,, otherFeat=[]-->, belongsTo=parr
954	2005). Thus, our findings that CFIm68 is released from the, otherFeat=[]-->, belongsTo=parr
955	mRNP at this stage may have interesting implications not, otherFeat=[]-->, belongsTo=parr
956	only for mRNA export and translation initiation but also for, otherFeat=[]-->, belongsTo=parr
957	mRNA quality control mechanisms. For example, the pres-, otherFeat=[]-->, belongsTo=parr
958	ence of CF Im bound downstream of a stop codon may, otherFeat=[]-->, belongsTo=parr
959	contribute to the definition of the proper termination codon, otherFeat=[]-->, belongsTo=parr
960	in metazoa. Further studies on the role of the factors that, otherFeat=[]-->, belongsTo=parr
961	bind the 3 end of the mRNA should provide new important, otherFeat=[]-->, belongsTo=parr
962	insight into the mechanism that defines premature stop, otherFeat=[]-->, belongsTo=parr
963	codons and leads to NMD., otherFeat=[]-->, belongsTo=parr
964	ACKNOWLEDGMENTS, otherFeat=[]-->, belongsTo=parr
965	We gratefully acknowledge M. Bianchi (San Raffaele University, Milan, Italy),, otherFeat=[]-->, belongsTo=parrnote
966	J. Lykke-Andersen (University of Colorado, Boulder, CO), W.Y. Tarn (Aca-, otherFeat=[]-->, belongsTo=parrnote
967	demia Sinica, Taipei, Taiwan), R. Reed (Harvard Medical School, Boston, MA), otherFeat=[]-->, belongsTo=parrnote
968	and S. Wilson (University of Sheffield, Sheffield, United Kingdom) for plas-, otherFeat=[]-->, belongsTo=parrnote
969	mids; F. Loreni (University 'Tor Vergata,' Rome, Italy) for antibodies; E., otherFeat=[]-->, belongsTo=parrnote
970	Izuarralde (Max Planck Institute for Developmental Biology, Tu?bingen, Ger-, otherFeat=[]-->, belongsTo=parrnote
971	many) for plasmids and antibodies; W. Keller (University of Basel, Basel,, otherFeat=[]-->, belongsTo=parrnote
972	Switzerland) for histidine-tagged CF Im subunits; and S. Stamm (University of, otherFeat=[]-->, belongsTo=parrnote
973	Erlangen, Erlangen, Germany) for Sf9 cells expressing His-Tra2 . We also, otherFeat=[]-->, belongsTo=parrnote
974	thank A. I. Lamond, J. Sanford, and J. Caceres for help with some of the, otherFeat=[]-->, belongsTo=parrnote
975	experiments and O. Mu?hlemann for valuable suggestions and proofreading, otherFeat=[]-->, belongsTo=parrnote
976	of the manuscript. We appreciate the technical help of N. Kleinschmidt, Lukas, otherFeat=[]-->, belongsTo=parrnote
977	Stalder, Sandro Waltersperger, and K. Schranz. This study was partially, otherFeat=[]-->, belongsTo=parrnote
978	supported by a MIUR-PRIN 2006 grant and by the Cariplo Foundation. S.C., otherFeat=[]-->, belongsTo=parrnote
979	was supported by a short-term EMBO fellowship. M-D.R. and D.S. were, otherFeat=[]-->, belongsTo=parrnote
980	supported by the Swiss National Science Foundation (Grants 3100A0-105547, otherFeat=[]-->, belongsTo=parrnote
981	and -120064) and the Canton Bern., otherFeat=[]-->, belongsTo=parrnote
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994	mRNA 3 end processing factor mammalian cleavage factor I 68-kDa subunit., otherFeat=[]-->, belongsTo=parrnote
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==============================
0	Molecular Biology of the Cell-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	Molecular Biology of>>> Biology of the Cell
1	Vol. 20, 5211­5223, December 15, 2009-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	Vol. 20, 5211­5223, >>>3, December 15, 2009
2	Mammalian pre-mRNA 3 End Processing Factor CF Im68-->id=3, page=0, size=15, fam=Times, col=#231f20, type=title, textLines=1--->[]--->note	Mammalian pre-mRNA 3>>>ssing Factor CF Im68
3	Functions in mRNA Export-->id=1, page=0, size=25, fam=Times, col=#231f20, type=title, textLines=1--->[]--->title	Functions in mRNA Ex>>>tions in mRNA Export
4	Marc-David Ruepp,* Chiara Aringhieri, Silvia Vivarelli, Stefano Cardinale,§ Simona Paro, Daniel Schu¨mperli,* and Silvia M.L. Barabino-->id=4, page=0, size=17, fam=Times, col=#231f20, type=title, textLines=2--->[]--->note	Marc-David Ruepp,* C>>>Silvia M.L. Barabino
5	Department of Biotechnology and Biosciences, University of Milano-Bicocca, I-20126 Milano, Italy; and-->id=7, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=2--->[]--->note	Department of Biotec>>>6 Milano, Italy; and
6	*Institute of Cell Biology, University of Bern, CH-3012 Bern, Switzerland-->id=7, page=0, size=13, fam=Times, col=#231f20, type=title, textLines=2--->[]--->note	*Institute of Cell B>>>12 Bern, Switzerland
7	Submitted May 13, 2009; Revised October 14, 2009; Accepted October 19, 2009-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	Submitted May 13, 20>>>ted October 19, 2009
8	Monitoring Editor: Marvin Wickens-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	Monitoring Editor: M>>>itor: Marvin Wickens
9	Export of mRNA from the nucleus is linked to proper processing and packaging into ribonucleoprotein complexes. Although several observations indicate a coupling between mRNA 3 end formation and export, it is not known how these two processes are mechanistically connected. Here, we show that a subunit of the mammalian pre-mRNA 3 end processing complex, CF Im68, stimulates mRNA export. CF Im68 shuttles between the nucleus and the cytoplasm in a transcription-dependent manner and interacts with the mRNA export receptor NXF1/TAP. Consistent with the idea that CF Im68 may act as a novel adaptor for NXF1/TAP, we show that CF Im68 promotes the export of a reporter mRNA as well as of endogenous mRNAs, whereas silencing by RNAi results in the accumulation of mRNAs in the nucleus. Moreover, CF Im68 associates with 80S ribosomes but not polysomes, suggesting that it is part of the mRNP that is remodeled in the cytoplasm during the initial stages of translation. These results reveal a novel function for the pre-mRNA 3 end processing factor CF Im68 in mRNA export.-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Export of mRNA from >>>Im68 in mRNA export.
10	INTRODUCTION-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	INTRODUCTION>>>INTRODUCTION
11	The removal of introns by splicing as well as cleavage and polyadenylation at the 3 end of RNA polymerase II primary transcripts (pre-mRNAs) are usually required before they can be exported from the nucleus as mature mRNAs (Erkmann and Kutay, 2004). This observation has suggested that transport factors interact with the RNA during premRNA processing. Indeed, recent discoveries have lent support to this hypothesis. The splicing reaction deposits on the mRNA a specific subset of proteins called the exon junction complex (EJC, for review see Tange et al., 2004). REF, a component of the EJC, facilitates mRNA export by interacting with the mRNA export factor NXF1 (also called TAP, for review, see Reed and Hurt, 2002). NXF1 was originally identified as the export receptor for type D retroviral RNAs that associate with NXF1 through a sequence-specific interaction with the constitutive transport element (CTE). However, NXF1 recruitment on cellular mRNAs requires adaptor proteins such as Aly/REF (hereafter named REF). In yeast Mex67 (the homolog of NXF1) is recruited by Yra1 (homolog of REF), which is also essential for the export of poly(A) RNA in Saccharomyces cerevisiae. In contrast in metazoans, REF is dispensable for bulk mRNA export. This raises the possibility that multiple and partially redundant adaptor proteins may be responsible for the recruitment of NXF1. Indeed, spliceosomal proteins, including U2AF35 (Zolotukhin et al., 2002) and some members of the SR family of splicing factors, were shown to interact with NXF1 and act as adaptors for NXF1-dependent export of poly(A) mRNAs (Huang and Steitz, 2001; Huang et al., 2003; Lai and Tarn, 2004; Hargous et al., 2006; Tintaru et al., 2007).-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	The removal of intro>>>ntaru et al., 2007).
12	Several observations have linked 3 end cleavage and polyadenylation to mRNA export (for review see Zhao et al., 1999). For example, RNA polymerase II reporter transcripts lacking a polyadenylation signal are retained in the nucleus of yeast cells. Positioning a transcribed poly(A) tract at the end of an mRNA by ribozyme cleavage does not result in efficient nuclear export, indicating that the 3 end processing reaction itself, and not simply the presence of the poly(A) tail, is required for nuclear export (Huang and Carmichael, 1996). Moreover, the NS1A protein of influenza A virus specifically inhibits export of cellular but not viral mRNAs by targeting two essential components of the pre-mRNA 3 end processing machinery (Nemeroff et al., 1998). Although three yeast polyadenylation factors, Hrp1/Nab4, Nab2, and Pab1 (Kessler et al., 1997; Hector et al., 2002; Brune et al., 2005), and the mammalian nuclear poly(A) tail­ binding protein (PABPN1, Calado et al., 2000), are nucleocytoplasmic shuttling proteins, to date there is no evidence for a direct role of 3 end processing factors in mRNA export.-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Several observations>>>tors in mRNA export.
13	The mature 3 ends of most eukaryotic mRNAs are generated by endonucleolytic cleavage of the primary transcript followed by the addition of a poly(A) tail to the upstream cleavage product (for reviews see Zhao et al., 1999; Gilmartin, 2005). Cleavage Factor Im (CF Im) is a component of the 3 end processing complex that participates in the cleavage reaction. CFIm is a heterodimer composed of a small subunit of 25 kDa and a large subunit of 59, 68, or 72 kDa (Ru¨egsegger et al., 1998). The 25- and 68-kDa subunits have been shown to be sufficient to reconstitute CFIm activity for poly(A) site cleavage in vitro upon addition to partially purified 3 processing factors (Ru¨egsegger et al., 1998). Both subunits contact the RNA substrate, as demonstrated by UV cross-linking studies (Ru¨gsegger et al., 1996). The structure of the CFIm 68-kDa subunit is strikingly similar to that of the SR family of proteins, which plays an essential role in basal and regulated pre-mRNA splicing (reviewed by Graveley, 2000). The 68-kDa protein possesses an N-terminal RNPtype RNA recognition motif (RRM) and an RS-like C-terminal region enriched in RS/D/E dipeptides that is reminiscent of the RS domain of SR proteins. The RS-like domain of CF Im 68-kDa subunit is sufficient for the localization in nuclear speckles (and in the nucleoplasm) and mediates the interaction in vitro with a subset of shuttling SR proteins (Dettwiler et al., 2004; Cardinale et al., 2007).-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	The mature 3 ends of>>>inale et al., 2007).
14	This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E09 ­ 05­ 0389) on October 28, 2009.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->parr	This article was pub>>>on October 28, 2009.
15	These authors contributed equally to this work.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->parr	These authors contri>>>qually to this work.
16	Present addresses: §Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Mailstop Stanley 922, Berkeley, CA 94720; Medical Research Council Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, United Kingdom.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->parr	Present addresses: §>>>2XU, United Kingdom.
17	Address correspondence to: Silvia M.L. Barabino: (silvia.barabino@ unimib.it).-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->parr	Address corresponden>>>arabino@ unimib.it).
18	© 2009 by The American Society for Cell Biology-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	© 2009 by The Americ>>>ety for Cell Biology
19	5211-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	5211>>>5211
20	Here, we demonstrate that CF Im68 is a nucleocytoplasmic shuttling protein that can stimulate mRNA export. First, we show that CF Im68 is associated with components of the EJC. Second, we establish that CF Im68 interacts with the mRNA export factor NXF1. Finally, we demonstrate that CF Im68 is directly involved in mRNA export. Although CF Im68 can stimulate export when tethered to a luciferase reporter mRNA and its overexpression results in an increase of endogenous mRNAs in the cytoplasmic fraction, RNAi-mediated silencing leads to the retention of polyadenylated transcripts in the nucleus. Furthermore, we show by sucrose gradient centrifugation that CF Im68 cosediments with the 80S ribosome particle. CF Im68 is a well-established component of the pre-mRNA 3 end processing complex. Our data showing that CF Im68 is part of the mature mRNP particles and contributes to their export to the cytoplasm highlight a novel function for CF Im68 and provide a link between pre-mRNA 3 end formation and mRNA export.-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Here, we demonstrate>>>ion and mRNA export.
21	MATERIALS AND METHODS-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->['U']--->title	MATERIALS AND METHOD>>>ATERIALS AND METHODS
22	Oligonucleotides, Plasmids, and Antibodies-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Oligonucleotides, Pl>>>mids, and Antibodies
23	Sequences for real-time RT-PCR probes and description of plasmids and antibodies are presented in Supplemental Material.-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	Sequences for real-t>>>pplemental Material.
24	Cell Culture and Heterokaryon Assay-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Cell Culture and Het>>>d Heterokaryon Assay
25	HeLa and HEK293 cells were cultured in DMEM supplemented with 10% FBS and transfected using Effectene Transfection Reagent (Qiagen, Chatsworth, CA) or Escort V (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions. Drug treatments were carried out as follows: actinomycin D treatment (5 g/ml, Sigma-Aldrich) for2hor leptomycin B (LMB; 25 g/ml, Sigma-Aldrich) for1hin DMEM, followed by fixation and fluorescence microscopy.-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	HeLa and HEK293 cell>>>rescence microscopy.
26	For heterokaryon nucleocytoplasmic shuttling assays, HeLa cells were transiently transfected with different vectors as described. Twenty-four hours after transfection, an equal number of HeLa cells was seeded onto the same coverslip as NIH3T3 cells. The cells were incubated for3hinthe presence of 50 g/ml cycloheximide and for 30 min in the presence of 100 g/ml cycloheximide before fusion. Cell fusions were done as described (PinolRoma and Dreyfuss, 1992). Heterokaryons were incubated further for2hin media containing 100 g/ml cycloheximide before fixation.-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	For heterokaryon nuc>>>ide before fixation.
27	Immunoprecipitations-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Immunoprecipitations>>>Immunoprecipitations
28	After transfection, HEK293 cells were washed and harvested. For the immunoprecipitations of Flag-hUpf3b complexes, 8 100-mm dishes of transfected cells were used for each assay. The pellets were quickly frozen in liquid nitrogen, thawed, and resuspended in lysis buffer (50 mM Tris HCl, pH 8, 150 mM NaCl, 1% NP40, and 1 protease inhibitor cocktail [PIC]; Roche, Indianapolis, IN) and then incubated on ice for 1 h with occasional shaking. For the coimmunoprecipitation of hemagglutinin (HA)-CF Im68 with either Flag-CF Im25 or Flag-NXF1, cells were lysed in high-salt lysis buffer (50 mM Tris-HCl, pH 8.0, 250 mM NaCl, 1% NP40). The samples were then centrifuged at 15,000 rcf for 15 min at 4°C, and the supernatants were collected. RNase A digestion (200 mg/ml, Roche) was performed for 30 min at 30°C.-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	After transfection, >>> for 30 min at 30°C.
29	Flag-tagged proteins were immunoprecipitated from the precleared, RNase A­treated cell lysate with M2 anti-Flag agarose (Sigma-Aldrich) at 4°C for 2 h on a rotator wheel in IP150 buffer (10 mM MgCl2, 10% NP40, 150 mM NaCl). Then samples were centrifuged ten times at 600 g for 10 min at 4°C and washed five times with 500 l of IP150 or IP250 buffer. Precipitated proteins were eluted either with SDS sample buffer or M2 peptide and analyzed by Western blotting. Detection was performed with an ECL detection kit (Amersham, Piscataway, NJ).-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	Flag-tagged proteins>>>am, Piscataway, NJ).
30	GST-Fusion Protein Purification and GST-Pulldown Assays-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	GST-Fusion Protein P>>> GST-Pulldown Assays
31	To study protein­protein interactions in vitro, GST fusion proteins were expressed in Escherichia coli BL21(DE3)LysS or BL21(DE3) RIPL transformed with pGEX-derived plasmids encoding glutathione S-transferase (GST; negative control) or GST-fusions with CFIm25, NXF1, or NXF1­202. After purification over glutathione Sepharose 4B beads (GE Healthcare, Waukesha, WI), GST-CFIm25 and GST-NXF1 were further purified by gel filtration over a Superdex 75 column (GE Healthcare).-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	To study protein­pro>>>umn (GE Healthcare).
32	GST pulldown assays with hexahistidine-tagged CF Im68 were performed as follows. The purified GST fusions were coupled to glutathione Sepharose 4B beads, by using equimolar amounts of either GST-CF Im25 or GST-NXF1 (1.5 and 3 g, respectively) and 3 g of GST as negative control. The beads and proteins were incubated in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,2mMKH2PO4) supplemented with 0.1% NP40 (PBS/NP40) at 4°C for 1.5 h with gentle agitation on a wheel, followed by the addition of 400 ng histidine-tagged CF Im68 and incubation for 1.5 h at 4°C. Subsequently, the beads were washed with PBS/NP40, and the bound material was analyzed by SDS-PAGE and subsequent Western blotting. Because input one-fifth of the added recombinant CF Im68 was loaded on the gel, His-CF Im68 was detected by incubation of the blot with a mouse monoclonal anti-His antibody (HIS1, Sigma-Aldrich) and a speciesspecific horseradish peroxidase­ coupled antibody (Promega, Madison, WI) and developed by the enhanced chemiluminescence method (ECL-Plus, GE Healthcare). To visualize the bound recombinant GST-fusions, the membrane was stained with Coomassie Brilliant Blue R-250.-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	GST pulldown assays >>>rilliant Blue R-250.
33	GST pulldown assays with in vitro­translated proteins were performed as follows. The purified GST-fusions were coupled to glutathione Sepharose 4B beads (GE Healthcare) in PBS/NP40 for 1 h and subsequently incubated with RNAse treated [35S]methionine-labeled proteins obtained by coupled in vitro transcription/translation in rabbit reticulocyte lysate (TNT T7 kit, Promega). For nonradioactive in vitro translation, methionine was added to a final concentration of 20 ­30 M. The samples were incubated in PBS supplemented with 0.1% NP40 at 4°C for 2 h with gentle agitation. Subsequently beads were washed with PBS/NP40 (NP40 concentration increased to 1%), and input and the bound fraction were analyzed by SDS-PAGE and detected on a storm 820 Phosphorimager (Amersham).-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	GST pulldown assays >>>orimager (Amersham).
34	Tethered mRNA Export Assay 0.3% NP40 for 2 min at 72°C, once with 2 SSC 0.1% NP40 for 1 min at room temperature (RT), and once with 2 SSC for 10 min at RT). After a preincubation wash with 4 SSC 0.1% Triton X-100 for 5 min at RT, the FISH probe was revealed with 3 g/ml streptavidin-APC conjugate (BD Biosciences, San Diego, CA) diluted in 4 SSC, 1% BSA for1hatRT. Finally, cells were stained with DAPI and mounted with FluorSave reagent (Calbiochem, La Jolla, CA). Images were collected with a TCS SP2 AOBS confocal microscope (Leica Microsystems, Exton, PA) and by using LSC software. Twelve-bit images were acquired by using the same setting parameters for all the samples (gain, offset); for each field, five different xy sections along the z-axis were acquired. FISH quantification was carried out with the same LCS software. Measurements of the FISH probe were obtained for the nuclear fluorescence (Sn), the total cell fluorescence (Sc), the area of the nucleus (An), and area of the cell (Ac). The cytoplasmic (C ) amount of mRNA was calculated as follows: C 1 [(An Sn)/(Ac Sc)]. C/N ratios were calculated as C/N C /(1 C ) (for reference see Valencia et al., 2008).-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Tethered mRNA Export>>>encia et al., 2008).
35	Assays were carried out as described in Hargous et al. (2006) and Tintaru et al. (2007), with the exception that renilla luciferase rather than ß-galactosidase was used for the normalization of transfection efficiency. For each transfection, 700 ng of each of the plasmids encoding the MS2-protein, 50 ng of luc-RRE firefly construct, and 5 ng of pRL-TK, a thymidine kinase renilla luciferase control vector, were cotransfected in 24-well plates. Detection of luciferase activity was performed with the Dual-luciferase Reporter Assay System (Promega) according to the manufacturer's instruction. Luminescence measurements were performed by using a Berthold luminometer. Four independent sets of transfections were carried out in triplicate with two different plasmids preparations. The average normalized luciferase activity in all the experiments was calculated and expressed as percentage of the activity measured for REF.-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	Assays were carried >>>ty measured for REF.
36	For analysis of the tethering experiments on the RNA-level, 1.4 106 HeLa cells were transfected with 10 g MS2 fusion plasmid and 500 ng of pLucSalRRE-6MS2 using Dreamfect (OZ Biosciences, Marseilles, France). The cells were harvested 48 h after transfection. Nuclei were isolated as described below and RNA was prepared by using an "Absolutely RNA RT-PCR Miniprep Kit" (Stratagene, La Jolla, CA). RNA, 1 g, was reverse-transcribed with random hexamers and StrataScript 6.0 reverse transcriptase (Stratagene) according to the manufacturer's protocol. Real-time RT-PCR was performed as described below.-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	For analysis of the >>> as described below.
37	Fluorescent In Situ Hybridization-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Fluorescent In Situ >>>n Situ Hybridization
38	For the visualization of the luciferase reporter RNA, the fluorescent in situ hybridization (FISH) probes were 390 nt biotinylated antisense RNA molecules transcribed in vitro from pRRE-Luc linearized with EcoRV with the BioArray HighYield RNA Transcript Labeling Kit (Enzo Life Sciences, New York, NY). HeLa cells were transiently transfected with pLUCRRE6MS2 reporter alone or cotransfected with pCNMS2CFIm68GFP, pCNMS2GFP, pCNMS2TAP, pCNMS2REF, or pCNMS2REF-RRM. After 30 h, the cells were fixed and FISH was performed according to standard protocols. Briefly, cells were incubated in prehybridization buffer (2 SSC, 20% formamide, 0.2% BSA, 1 g/ l tRNA) for 30 min at 37°C and then in hybridization solution (2-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	For the visualizatio>>>dization solution (2
39	SSC, 20% formamide, 0.2% BSA, 10% dextran sulfate, 1 g/ l tRNA) in the-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	SSC, 20% formamide, >>> 1 g/ l tRNA) in the
40	M.-D. Ruepp et al.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	M.-D. Ruepp et al.>>>M.-D. Ruepp et al.
41	Molecular Biology of the Cell-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	Molecular Biology of>>> Biology of the Cell
42	5212-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	5212>>>5212
43	presence of the biotinylated RNA probe (50 ng/slide) for3hat 37°C. Stringent washes were performed in order to wash out unlabeled probe (twice with 2 SSC 20% formamide, twice with 2 SSC, once with 1 SSC for 15 min at 45°C, once with 0.5 SSC for 15 min at 45°C, once with 0.5 SSC-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	presence of the biot>>>C, once with 0.5 SSC
44	For the visualization of poly(A) RNA, HeLa cells were grown in six-well plates on coverslips and transfected with the different plasmids, as indicated. Seven days past transfection, the cells were washed two times in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4,2mMKH2PO4) before they were fixed for 10 min at RT in 3.7% paraformaldehyde (PFA). After a wash in PBS for 5 min, the cells were permeabilized for 10 min with 100% methanol, followed by a 10-min incubation in 70% ethanol, and a 5-min incubation in 1 M Tris, pH 8.0. The cells were then blocked for 30 min at 40°C in prewarmed prehybridization solution (2 x SSC, 20% formamide, 0.2% BSA, 1 g/ l tRNA in DEPC-H2O). Prewarmed hybridization solution (2 SSC, 20% formamide, 10% dextran sulfate, 0.2% BSA, 1 g/ l tRNA, 1 ng/ l Cy3-labeled oligo(dT)50 in DEPC-H2O) was added to the coverslips and incubated at 37°C for 2 h. Next, the cells were washed twice with prewarmed 2 SSC and 20% formamide in PBS, twice with prewarmed 2 SSC in PBS for 5 min each at 42°C and once in 1 SSC in PBS for 5 min at RT. Before the coverslips were mounted (Mowiol 4 ­ 88, Calbiochem, containing 0.25 g DAPI/ml), they were washed three times for 5 min in PBS.-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	For the visualizatio>>>es for 5 min in PBS.
45	RNA Interference-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	RNA Interference>>>RNA Interference
46	HeLa cells stably transfected with pcDNA3-ß-globin cDNA were transfected either with two siRNAs against CF Im68 or NXF1 (Silencer Select Pre-Designed & Validated siRNA, Applied Biosystems, Foster City, CA) at 25 nM concentration each or a nontargeting siRNA control at 50 nM concentration, by using Lullaby reagent (OZ Biosciences) according to the manufacturer's instructions. After splitting, cells were subjected to a second round of transfection with 50% of the amount of siRNAs indicated above. Cells depleted from CF Im68 were harvested 4 d after the first transfection for analysis, whereas cells depleted from NXF1 were harvested 48 h after transfection.-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	HeLa cells stably tr>>> after transfection.
47	Real-Time PCR Analysis-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Real-Time PCR Analys>>>al-Time PCR Analysis
48	For the analysis of the nuclear and cytoplasmic distribution of mRNAs, HeLa cells were cotransfected with pSUPuro constructs or pcDNA3-HA­ derived plasmids containing a puromycin selection marker by using Dreamfect (Oz Biosciences). Culturing the cells in the presence of 1.5 g/ml puromycin eliminated untransfected cells. Three days after transfection cells were harvested.-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	For the analysis of >>>ells were harvested.
49	Nuclear-cytoplasmic fractionations of HeLa cells were performed as described elsewhere (Carneiro and Schibler, 1984), except that RNase A treatment was omitted. From each fraction, 20 l aliquots were taken for Western blot analysis. Subsequently RNA was isolated by using TRI-Reagent (Ambion, Applied Biosystems, Inc., Foster City, CA), and DNA contamination was removed by using Turbo DNA-free (Ambion). Total RNA, 2 g, from each fraction was reverse transcribed with Stratascript reverse transcriptase (Stratagene) according to the manufacturer's instruction. cDNA corresponding to 40 ng RNA was amplified with specific primers and TaqMan probes in an ABI SDS7000 Sequence Detection System (Applied Biosystems). The relative mRNA levels of the cytoplasmic and nuclear fractions were normalized to 18S rRNA levels. The RT-PCR for X-ist and MIC 2a was performed in 50 l 1 Fast Start Master Mix (Roche) supplemented with 400 nM f.c. of each primer pair, and 38 cycles were performed.-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	Nuclear-cytoplasmic >>>cles were performed.
50	Polysome Profile-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Polysome Profile>>>Polysome Profile
51	Sucrose gradient fractionation was performed as described in Sanford et al. (2004). Briefly, HEK293 cells were cotransfected with plasmids expressing HA-CF Im68 and T7-ASF. After 24 h cells were washed and collected by short centrifugation at 4,000 rpm at 4°C and resuspended cold lysis buffer (100 mM NaCl, 10 mM MgCl2, 30 mM Tris HCl, pH 7.5, 1 mM DTT, 0.05% Triton X-100) containing 100 g/ml cycloheximide, RNase, and protease inhibitors. After 5 min on ice, the extract was centrifuged for 4 min at 12,000 g at 4°C to pellet nuclei and debris. For negative controls, extracts were treated before sucrose gradient analysis either with 30 mM EDTA for 10 min at 4°C on a rotator wheel, followed by the addition of 2 g RNase A (Roche) and further incubation for 30 min at 30°C, or with 20 mM puromycin for1hat4°C on a rotator wheel. Supernatants were resolved on 15­50% sucrose gradients prepared in (100 mM NaCl, 10 mM MgCl2, 10 mM Tris HCl, pH 7.5). The gradient was centrifuged at 4°C at 38,000 rpm in a Beckman SW41 rotor for 3 h. After centrifugation, fractions were collected from the bottom, precipitated with TCA, and analyzed by immunoblotting.-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	Sucrose gradient fra>>>d by immunoblotting.
52	Online Supplemental Materials are as follows: Supplemental File 1: legends to Supplemental Figures S1­S8; Supplemental File 2: Supplemental methods; Supplemental Figure S1: CF Im is imported into the nucleus as a heterodimer; Supplemental Figure S2: Nucleocytoplasmic shuttling is an active process; Supplemental Figure S3: Western blot analysis of HEK293 cell extracts used in the mRNA export assay; Supplemental Figure S4: Western blot assessing the purity of the nuclear fractions used in the experiments shown in Figure 6D and 7E; Supplemental Figure S5: NXF1 depletion increases nuclear levels of ß-globin mRNA; Supplemental Figure S6: sucrose gradient fractionation of puromycin-treated extracts; Supplemental Figure S7: color version of Figure 1; and Supplemental Figure S8: color version of Figure 2.-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	Online Supplemental >>>version of Figure 2.
53	RESULTS-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	RESULTS>>>RESULTS
54	CF I m Large Subunits Are Nucleocytoplasmic Shuttling Proteins-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	CF I m Large Subunit>>>c Shuttling Proteins
55	We reported previously that the 68-kDa subunit of CF Im interacts with a subset of SR proteins that were shown to shuttle continuously between the nucleus and the cytoplasm (Dettwiler et al., 2004). In addition, the 25-kDa subunit interacts with PABPN1, which is a nucleocytoplasmic shuttling protein (Calado et al., 2000). On the basis of these observations we wanted to test whether also the CF Im subunits shuttle between the nucleus and the cytoplasm. We analyzed their migration in an interspecies heterokaryon fusion assay. HeLa cells expressing HA-tagged CF Im68 were fused to mouse NIH3T3 cells in the presence of cycloheximide to produce heterokaryons. Shuttling of HA-CF Im68 would lead to its equilibration into the nuclei of fused NIH3T3 cells. As a control, HeLa cells were cotransfected with a plasmid expressing GFP-hnRNP A1, a well-known shuttling protein, or GFP-hnRNP C, a protein that is always restricted to the nucleus (Pinol-Roma et al., 1988). In a representative heterokaryon, 2 h after fusion, HA-CF Im68 and GFP-hnRNP A1 were present both in the two HeLa cell nuclei and in the mouse cell nucleus (Figure 1A, top row). In contrast, GFP­ hnRNP C was restricted, as expected, to the HeLa cell nucleus (Figure 1A, bottom row). Quantitative analysis showed that NIH3T3 nuclei were positive for HA-CF Im68 in all of the 51 heterokaryons examined. Thus, although CF Im68 is nuclear at steady state, it is continuously traversing the nuclear envelope. A similar assay was performed with HeLa cells expressing either GFP-CF Im59 or GFP-CF Im25. As shown in Figure 1B, although CF Im59 can efficiently migrate into the mouse nucleus (Figure 1B, top row), shuttling of CF Im25 is less efficient (Figure 1B, bottom row). A possible explanation for the observed, inefficient migration of CF Im25 into the mouse nucleus could be that this subunit is not able to shuttle on its own and may be imported into the nucleus only in association with one of the endogenous larger polypeptides. Therefore, if GFP-CF Im25 is overexpressed but the endogenous large subunits are limiting, this may result in poor nuclear import of GFP-CF Im25. Cotransfection experiments with CFP-CF Im68 and YFP-CF Im25 constructs in which both ORFs are expressed under the same strong promoter (so that the two CF Im subunits should be present in the cell in almost equimolar amounts and could therefore efficiently dimerize) demonstrated that, under these conditions, the CF Im 25-kDa subunit is imported into nucleus more efficiently, in agreement with the explanation proposed above (Supplemental Figure S1).-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	We reported previous>>>lemental Figure S1).
56	To identify the region involved in the export of CF Im68, we analyzed the shuttling behavior of various domain deletion mutants fused to GFP (68 N, 68 RS, 68RS, and 68RRM/RS, depicted in Supplemental Figure S2; Dettwiler et al., 2004) at 37°C or, in addition, at 4°C to check for passive diffusion. The rationale for this assay is that, at 4°C, both receptor-mediated nuclear import and export are blocked, whereas passive diffusion continues to occur. Temperatureshift experiments were performed as described in Michael et al. (1995). As shown in Supplemental Figure S2, all the mutants were able to shuttle at 37°C but were restricted to the HeLa cell nucleus at 4°C.-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	To identify the regi>>>cell nucleus at 4°C.
57	Nucleocytoplasmic Shuttling of CF Im-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	Nucleocytoplasmic Sh>>>c Shuttling of CF Im
58	Vol. 20, December 15, 2009-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	Vol. 20, December 15>>>0, December 15, 2009
59	5213-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	5213>>>5213
60	Nuclear Export of CF Im68 is CRM1-independent and Requires Active Transcription-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Nuclear Export of CF>>>Active Transcription
61	The most common mechanism for the nuclear export of proteins in eukaryotic cells is based on CRM1-dependent systems. CRM1-mediated export is specifically blocked in the presence of the fungal metabolite LMB, which inhibits the formation of the ternary complex between CRM1, RanGTP, and the cargo protein. To check whether CRM1 is responsible for CF Im68 nuclear export, heterokaryon assays were thus performed the presence of LMB. HeLa cells treated in this way were transfected with plasmids expressing HA-CF Im68 in combination with GFP-HMGB1, a protein that requires CRM1 for export (Figure 2A). Treatment with LMB specifically restricted HMGB1 to the HeLa cell nucleus. while CF Im68 could still be efficiently exported thus indicating that its export does not require CRM1.-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	The most common mech>>>es not require CRM1.
62	Previous studies have shown that CF Im is an RNA-binding factor (Ru¨egsegger et al., 1996). CF Im68 contacts the RNA mainly via the charged C-terminal domain, whereas the RRM is primarily involved in protein interactions with the small subunit (Dettwiler et al., 2004). Therefore, it is possible that CF Im68 is leaving the nucleus by "piggy-backing" on RNA molecules being exported by other factors, either in association with the 25-kDa subunit or because of a direct interaction via the C-terminal domain. To determine whether shuttling of CF Im68 depends on mRNA synthesis, we performed heterokaryon assays in the presence of actinomycin D. As shown in Figure 2B, GFP-CF Im68 shuttling is blocked in presence of the transcription inhibitor, whereas relocalization of the protein in cap-like structures around the nucleoli of the human cell can be observed, as previously described (Cardinale et al., 2007; Figure 2B). Although we cannot formally rule out the possibility that actinomycin D could affect the synthesis of a short-lived protein required for export, the most likely conclusion of these experiments is that CF Im68 shuttling is dependent on mRNA synthesis and possibly export. Therefore, on the basis of this observation, we began to investigate a possible involvement of CF Im68 in mRNA export.-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Previous studies hav>>>Im68 in mRNA export.
63	Figure 1. The two large CF Im subunits migrate between nuclei in interspecies heterokaryons. (A) CF Im68 is a shuttling protein. Left, merge of DAPI staining of HeLa and NIH3T3 nuclei (indicated by broken arrows) and phase-contrast microscopy view. Middle, localization of GFP­ hnRNP A1 or GFP--->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->capfig	Figure 1. The two la>>>FP­ hnRNP A1 or GFP-
64	hnRNP C. Right, localization of HA-CF Im68.-->id=14, page=0, size=5, fam=Times, col=#231f20, type=note, textLines=4--->[]--->capfig	hnRNP C. Right, loca>>>ation of HA-CF Im68.
65	(B) CF Im59 but not the 25-kDa subunit shuttles between the nucleus and the cytoplasm. Representative heterokaryons of HeLa cells, transfected with either GFP-CF Im59 or GFP-CF Im25. Broken arrows, the mouse nuclei. A full-color version of this figure is available as Supplementary Figure S7.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->capfig	(B) CF Im59 but not >>>lementary Figure S7.
66	Figure 2. Export of CF Im68 depends on mRNA traffic. (A) Inhibition of the CRM1-mediated nuclear export pathway does not affect CF Im68-->id=14, page=0, size=5, fam=Times, col=#231f20, type=note, textLines=4--->[]--->capfig	Figure 2. Export of >>>s not affect CF Im68
67	shuttling activity. HeLa cells were cotransfected with expression constructs for HA-CF Im68 and GFP-HMGB1. Two hours before fusion with NHI3T3 cells and throughout the experiment, cells were incubated in the absence (not shown) or in the presence of LMB as described in Materials and Methods. HA-CF Im68 was detected by immunofluorescence. (B) HeLa cells transfected with GFP-CF Im68 and mouse NIH3T3 cells were treated with actinomycin D and then fused as described in Materials and Methods. Left, merge of DAPI staining of HeLa and NIH3T3 nuclei (indicated by broken arrows) and phase-contrast microscopy view. A full-color version of this figure is available as Supplementary Figure S8.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->capfig	shuttling activity. >>>lementary Figure S8.
68	M.-D. Ruepp et al.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	M.-D. Ruepp et al.>>>M.-D. Ruepp et al.
69	Molecular Biology of the Cell-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	Molecular Biology of>>> Biology of the Cell
70	5214-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	5214>>>5214
71	CF I m68 Interacts with the mRNA Export Factor NXF1-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	CF I m68 Interacts w>>>A Export Factor NXF1
72	The EJC, which is deposited on the mRNA by the splicing reaction, consists of four core components (Y14, Magoh, MLN51, and eIFAIII) and several more peripherally associated proteins (for review see Tange et al., 2004). Complexed proteins include the splicing factors SRm160, RNPS1, Acinus, SAP18 and Pinin, the mRNA export factors UAP56, Aly/REF, and NXF1, and the NMD factor Upf3b. To determine if CF Im68 may be present in postsplicing complexes, Upf3b-containing mRNPs were immunopurified from extracts of HEK293 cells transiently transfected with Flag­ tagged hUpf3b and analyzed by Western blotting with antibodies against EJC proteins, hnRNP proteins, and with anti-CF Im68 antiserum (Figure 3A). Because several of these factors, including CF Im, can bind RNA, extracts were treated with RNase A before immunoprecipitation to test for true protein­protein interactions. RNase treatment was shown to be effective by the disappearance of ß-actin mRNA as revealed by RT-PCR (Figure 3A, left). Consistent with previous reports, CBP80, NXF1, REF, and Magoh coimmunopurified with Flag-hUpf3b (Figure 3A, right; Kim et al., 2001; Lejeune et al., 2002; Singh et al., 2007). In addition to these proteins, CF Im68 can also be detected in Upf3b-containing mRNPs even in the presence of RNaseA, suggesting that it must interact with at least one of the non-RNA components of the complex. As expected, hnRNPA1, which is an abundant component of heterogeneous nuclear RNP (hnRNP) complexes but not of postsplicing complexes (Kim et al., 2001), is not present in the pellet of the coimmunoprecipitation.-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	The EJC, which is de>>>immunoprecipitation.
73	Because export of CF Im68 is not mediated by CRM1 but instead requires mRNA transcription, we asked whether NXF1 might be the export receptor for CF Im68. Control Flag-tagged CF Im25, -NXF1, -Upf1, -Upf3b, or the empty Flag vector alone were transiently coexpressed with HA--->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Because export of CF>>>coexpressed with HA-
74	tagged CF Im68 in HEK293 cells. Interaction with CF Im68-->id=10, page=0, size=6, fam=Times, col=#231f20, type=parrnote, textLines=20--->[]--->parr	tagged CF Im68 in HE>>>raction with CF Im68
75	was analyzed by immunoprecipitation of the cell lysates with anti-Flag antibodies in the presence of RNase A followed by Western blotting with anti-CF Im68 antiserum. The expression level of Flag-NXF1 was significantly lower than that of the other Flag-tagged proteins, and especially of Flag-Upf1 (Figure 3B, input, cf. lanes 4 and 5). Nevertheless, the amount of CF Im68 associated with Flag-NXF1 was far greater than that obtained with either Flag-Upf3b or Flag alone, and equivalent to the amount coprecipitated with Flag-Upf1 (pellet, cf. lanes 8 ­11). These results suggest that CF Im68 interacts specifically with NXF1 in vivo, and possibly also with Upf1. Thus, to determine if the interaction between CF Im68 and NXF1 is a direct one, we performed GST pulldown experiments with purified recombinant proteins. E. coli­ expressed GST, control GST-CF Im25, or GSTNXF1 were incubated with purified baculovirus-expressed, histidine-tagged CF Im68. Bound proteins were analyzed by Western blotting using anti-histidine (Figure 3C). Because GST­NXF1 coprecipitated equivalent amount of CF Im68 to that coprecipitated by CF Im25 (cf. lanes 2 and 3), we conclude that CF Im68 interacts specifically with the mRNA export factor NXF1.-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	was analyzed by immu>>> export factor NXF1.
76	We next aimed to identify the region in CF Im68 responsible for the interaction with NXF1. We tested the interaction by coimmunoprecipitation of Flag-tagged NXF1 with GFP fusions of wild-type and mutant CF Im68 that were coexpressed in HEK293 cells (Figure 4B). Tested mutants included a deletion of the N-terminal portion of CFIm68, a region that contains the RRM and was shown to bind the 25-kDa subunit (68 N), a deletion of the entire RS-like region at the C-terminus (68 RS), the RS-like domain alone (68RS), and a fusion of the RRM and the RS domains (68RRM/RS). As shown in Figure 4B Flag-NXF1 precipitated the fragments 68RRM/RS and 68 RS. Deletion of the N-terminal portion impaired the interaction with NXF1, whereas the RS-like domain appeared to interact with NXF1 albeit very inefficiently. Similar results were obtained in GST pulldown experiments in which recombinant GST-CF Im68 truncations were incubated with Flag-NXF1 expressed in HEK293 cells (data not shown).-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	We next aimed to ide>>>ls (data not shown).
77	Figure 3. CF Im68 and NXF1 interact in HEK293 cells and in vitro. (A) CF Im68 associates with hUpf3b-containing protein complexes. Left, RTPCR quantitation of the level of ß-actin mRNA before or after RNase treatment. Extracts were treated with increasing amounts of RNaseA. Lane 1, untreated extract; lane 2, 1 g/ml; lane 3, 3 g/ml; lane 4, 15 g/ml. Right, coimmunoprecipitation experiment of RNase A­treated extracts of HEK293 cells transfected with Flag-Upf3b by using anti-Flag antibodies (lanes 3 and 5). For mock transfection, the same amount of Flag-pCMVTag2 was used as negative control (lanes 2 and 4). Coimmunoprecipitated proteins were visualized by Western blotting with the indicated antibodies. Lane 1, total extract from untransfected cells. (B) CF Im68 interacts with NXF1 in HEK293 cell extract. Coimmunoprecipitations with anti-Flag antibody of RNase A­treated extracts of HEK293 cells transfected with Flag-tagged CF Im25 (positive control, lanes 3 and 8), NXF1 (lanes 4 and 9), hUpf1 (lanes 5 and 10), or hUpf3b (lanes 6 and 11), and HA-tagged CF Im68, as indicated. For mock transfections (lanes 2 and 7), the same amount of Flag-pCMVTag2 was used. Bound proteins (Pellet) were detected by Western blotting with anti-CF Im68 antiserum (top panel). To control for equal loading, the membrane was sequentially probed without stripping with anti-Flag and anti-actin antibodies (bottom panel). An-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->capfig	Figure 3. CF Im68 an>>>s (bottom panel). An
78	asterisk indicates residual signal of the anti-CF Im68 antibody. Lane 1, total extract from untransfected cells. (C) Recombinant CF Im68-->id=14, page=0, size=5, fam=Times, col=#231f20, type=note, textLines=4--->[]--->capfig	asterisk indicates r>>> Recombinant CF Im68
79	interacts with NXF1 in vitro. Left, Coomassie-stained SDS-PAGE of the purified recombinant proteins used in the pulldown assay. Right, GST (lane 2), GST-tagged CF Im25 (lane 3) and NXF1 (lane 4) were tested for interaction with histidine-tagged CF Im68. Eluted proteins were analyzed by Western blotting with anti-histidine antibody.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->capfig	interacts with NXF1 >>>-histidine antibody.
80	Nucleocytoplasmic Shuttling of CF Im-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	Nucleocytoplasmic Sh>>>c Shuttling of CF Im
81	Vol. 20, December 15, 2009-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	Vol. 20, December 15>>>0, December 15, 2009
82	5215-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	5215>>>5215
83	To confirm the requirement of the N-terminal region of CF Im68 for the direct interaction with NXF1, in vitro binding assays were performed. Glutathione beads prebound with GST or GST-NXF1 were incubated with 35S-labeled CF Im68 or 68 N. Bound fractions were resolved and visualized by autoradiography. As shown in Figure 4C, only the fulllength protein, but not the N-terminally deleted one, bound NXF1, indicating that the NXF1 interaction region lies within the first 213 amino acids of CF Im68.-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	To confirm the requi>>>no acids of CF Im68.
84	NXF1 can be functionally divided into three domains (Figure 5A; Izaurralde, 2002): the N-terminal half (aa 1-372) interacts with REF (Stutz et al., 2000); the region between aa 371 and 551 binds the essential export cofactor p15 (Katahira et al., 1999; Guzik et al., 2001; Izaurralde, 2002); and the C-terminal domain interacts with components of the NPC (Bachi et al., 2000). Shuttling SR proteins interact with the labeled, HA-tagged CF Im68 or 68 N. As shown in Figure 5B only full-length NXF1, but not the first 202 aa, which bind shuttling SR proteins (Huang et al., 2003 and data not shown), interacts with CF Im68.-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	NXF1 can be function>>>eracts with CF Im68.
85	Figure 4. CF Im68 interacts with NXF1 via its N terminus. (A) Schematic representation of the domain structure of CF Im68. The open and solid boxes indicate the regions of the protein present in each mutant relative to the 552 amino acid wild-type protein shown at the top. The connecting line indicates missing residues (222­ 419) in the RRM/RS protein. (B) HEK293 cells were transfected either with pCMVTag2-Flag or with pFlagNXF1 and plasmids expressing GFP-tagged full-length CF Im68 (lanes 2­ 4), or the domain deletion mutant proteins 68 N (lanes 5­7), 68RRM/RS (lanes 8 ­10), 68RS (lanes 11­13), and 68 RS (lanes 14 ­16). Total extracts were immunoprecipitated with anti-Flag antibody and analyzed by Western blotting. Lane 1, extract from untransfected cells. (C) E. coli­ expressed GST or GST-NXF1 prebound to beads were individually incubated with in vitro­translated, 35S-labeled HA-tagged CF Im68 or 68 N. Bound fractions (lanes 2, 3, 5, and 6) were subjected to SDS-PAGE and analyzed by autoradiography.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->capfig	Figure 4. CF Im68 in>>> by autoradiography.
86	Figure 5. CF Im68 and SR proteins do not bind the same region of NXF1. (A) NXF1 domain (top) organization with the 202­aa fragment (bottom). The regions interacting with the SR proteins, REF, p15, and the NPC are indicated. (B) SR proteins and CF Im68 do not bind to the same NXF1 region. Left, E. coli­ expressed GST, GST-NXF1, or GST-NXF1 202 were incu--->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->capfig	Figure 5. CF Im68 an>>>-NXF1 202 were incu-
87	bated with in vitro­translated, 35S-labeled-->id=14, page=0, size=5, fam=Times, col=#231f20, type=note, textLines=4--->[]--->capfig	bated with in vitro­>>>nslated, 35S-labeled
88	CF Im68. Bound fractions were analyzed by autoradiography. Left, Coomassiestained gel of the purified recombinant proteins. (C) SR proteins can bridge the interaction between NXF1 and CF Im68. E. coli­ expressed GST, or GST-NXF1 were incubated with in vitro­translated, 35S-labeled CF Im68 N in the presence of recombinant, histidine-tagged hTra2ß as described in Materials and Methods. Bound fractions were analyzed by autoradiography.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->capfig	CF Im68. Bound fract>>> by autoradiography.
89	M.-D. Ruepp et al.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	M.-D. Ruepp et al.>>>M.-D. Ruepp et al.
90	Molecular Biology of the Cell-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	Molecular Biology of>>> Biology of the Cell
91	5216-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	5216>>>5216
92	same domain of NXF1 that binds REF (Huang et al., 2003). To determine if CF Im68 and SR proteins bind the same domain of NXF1, glutathione beads loaded with GST or GST-NXF1-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	same domain of NXF1 >>>with GST or GST-NXF1
93	were individually incubated with in vitro­translated, 35S--->id=10, page=0, size=6, fam=Times, col=#231f20, type=parrnote, textLines=20--->[]--->parr	were individually in>>>tro­translated, 35S-
94	We previously demonstrated that the C-terminal charged domain of CF I m68 interacts with shuttling SR proteins (Dettwiler et al., 2004). Thus, we tested whether the C-terminal region of CF Im68 may mediate the formation of a ternary complex between SR proteins and NXF1. In the presence of the SR protein hTra2ß, GST-NXF1 can indeed efficiently select 35S-labeled 68 N, which on its own did not bind NXF1 (cf. Figures 4C and 5C).-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	We previously demons>>> Figures 4C and 5C).
95	CF I m68 Stimulates mRNA Export-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	CF I m68 Stimulates >>>imulates mRNA Export
96	To test if CF Im68 may contribute to general mRNA export through the interaction with the receptor protein NXF1 (as is the case for REF and some SR proteins), we used a recently described, tethered mRNA export assay (Hargous et al., 2006). In this assay, an mRNA export factor is expressed as a fusion protein with a bacteriophage MS2 coat protein tag and then artificially tethered to a reporter RNA containing the luciferase ORF and six MS2 coat protein­ binding sites within an inefficiently spliced intron (Figure 6A). The binding of the export factor leads to the nuclear export of the unspliced RNA that would otherwise be retained in the nucleus, resulting in the expression of luciferase activity. We therefore checked whether tethering CF Im68 protein in this way would promote export of the reporter RNA. Vectors expressing the different MS2 fusion proteins were transfected into HEK293 cells together with the luciferase reporter construct. Cotransfection of a third vector encoding renilla luciferase was used to control the transfection efficiency. As controls, MS2 fusions of GFP, NXF1, REF, and REF-RRM (aa 71­155; Hargous et al., 2006) were tested in the same experiments. As seen in Figure 6B, direct tethering of NXF1 resulted in a very strong luciferase activity. Importantly, tethering of CF Im68 led to a luciferase expression that was 10 times lower than that elicited by NXF1, but still higher than that of REF. As expected, REF-RRM did not stimulate export of the RNA Control experiments revealed-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	To test if CF Im68 m>>>experiments revealed
97	Figure 6. Tethering of CFIm68 leads to mRNA export. (A) Schematic of pRRE-Luc construct. The position of the FISH probe is indicated. (B) Normalized firefly/renilla luciferase activity generated by the MS2 fusion proteins in the tethered mRNA export assay. Error bars, SDs from four independent sets of assays. The averaged activity of each MS2fusion protein was expressed as a percentage of the average activity measured for MS2-REF calculated as described in Materials and Methods. RRE, transfection with pRRE-Luc without MS2 fusion protein. (C) FISH of HeLa cells cotransfected with pRRE-Luc and double-tagged GFP/ MS2 (a­a ), GFP/MS2­CF Im68 (b­ b ), or Myc/MS2-NXF1 (c and c ). DAPI staining was used to identify the cell nucleus (a­ c), localization of RRE-Luc mRNA in representative cells was visualized by FISH (a ­c ); GFP fluorescence was used to identify cells expressing the exogenous protein (a and b ). (D) The average cytoplasmic to nuclear (C/N) ratio for the RRE-Luc mRNA was determined by quantitative analysis of FISH experiments as shown in C for the indicated number of cells per construct. Error bars, SDs. (E) Real-time PCR analysis of the relative amount of RRE-Luc mRNA in the nuclear fractions of cells transfected with the indicated MS2-fusions constructs as determined by real-time RT-PCR. Three experiments were performed and average values are shown.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->capfig	Figure 6. Tethering >>>ge values are shown.
98	Nucleocytoplasmic Shuttling of CF Im-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	Nucleocytoplasmic Sh>>>c Shuttling of CF Im
99	Vol. 20, December 15, 2009-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	Vol. 20, December 15>>>0, December 15, 2009
100	5217-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	5217>>>5217
101	Figure 7. CF Im68 promotes export of mRNAs. (A) Hela cells growing on coverslips, overexpressing HA-tagged CFIm68 and a puromycin resistance as well as control cells expressing only the puromycin resistance were fixed with formaldehyde and permeabilized with methanol. Polyadenylated mRNA was visualized by hybridization of a Cy3-labeled oligo(dT)50 probe, whereas the nuclei were stained with DAPI. The panels are projections of Z-stacks from top to bottom of the nuclei. Images were collected on a Leica TCS SP2 AOBS laser scanning confocal microscope equipped with a HCX PL APOlbd.BL 63.0 1.2W objective (Leica Microsystems). (B) PCR analysis of nuclear and cytoplasmic RNAs of the nuclear X-ist mRNA (top) and the X-encoded Mic 2A transcript (bottom). (C) Overxpression of CF Im68 stimulates mRNA export. Real-time PCR analysis of the relative amount of ß-actin, Gapdh, Upf2, and Smg7 mRNA levels in the cytoplasmic (top histogram) and nuclear (bottom histogram) fraction of cells expressing HA-tagged EGFP ( ) or HA-tagged CFIm68 (u). The values were normalized to 18S rRNA levels and then divided by the values obtained in cells expressing the EGFP control. Three independent experiments were performed and average values of three real-time PCR runs with cDNAs from one representative experiment are shown. (D) HeLa cells stably expressing ß-globin mRNA were transfected with a control siRNA or with siRNAs against CFIm68. Total extract was prepared and analyzed by Western blotting with anti-CFIm68 and with anti-SmB/B antibodies. Three different experiments are shown. (E) CFIm68 depletion increases nuclear levels of ß-globin mRNA. Real-time RT-PCR analysis of the relative amount of ß-globin mRNA in the nuclear fraction of HeLa cells transfected with a control siRNA or cells depleted of CFIm68. The indicated values are normalized to 18S rRNA levels. Three experiments were performed and average values are shown. (F) CFIm68 depletion does not affect pre-mRNA 3 end processing. Real-time RT-PCR of ß-actin mRNA was performed with probes spanning the 5 UTR or the cleavage site to measure total and unprocessed mRNA, respectively. The ratio of precursor to total mRNA (pre/tot) was calculated to assess the efficiency of 3 end processing. The processing activity of cells transfected with the control siRNA was set to 1.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->capfig	Figure 7. CF Im68 pr>>> siRNA was set to 1.
102	M.-D. Ruepp et al.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	M.-D. Ruepp et al.>>>M.-D. Ruepp et al.
103	Molecular Biology of the Cell-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	Molecular Biology of>>> Biology of the Cell
104	5218-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	5218>>>5218
105	that all the MS2 fusion proteins were expressed (Western blot; Supplemental Figure S3) and correctly localized to the cell nucleus (immunofluorescence microscopy, data not shown).-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	that all the MS2 fus>>>py, data not shown).
106	Mutant CF Im68 proteins were also tested by this assay. Deletion of the N-terminal domain (68 N) that is necessary for the direct interaction with NXF1 in vitro did not significantly affect mRNA export, most likely because the protein can still interact with NXF1 via SR protein(s) bound to the C-terminus (see above). In contrast, the deletion of 2/3 of the C-terminal RS motif (68 C) strongly reduced luciferase expression. However, because this fragment is known to be imported into the nucleus inefficiently (Dettwiler et al., 2004), this result must be interpreted with caution. In further control experiments, the 30- and the 73-kDa subunits of the cleavage and polyadenylation specificity factor CPSF had no effect when tethered to the reporter RNA (Figure 6B and data not shown, respectively).-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Mutant CF Im68 prote>>>hown, respectively).
107	The assay described above relies on the measurement of luciferase enzymatic activity and thus requires mRNA translation. To rule out that CF Im68 might affect the translation efficiency rather than RNA export, we sought to measure directly nuclear and cytoplasmic mRNA levels in the presence of varying amounts of CF Im68. First, steady-state pRRE-Luc mRNA levels were visualized by FISH; Figure 6C). Quantitation of FISH images for individual cells revealed that the cytoplasmic to nuclear (C/N) ratio of the FISH signal was 1 for the GFP control, 2.28 for GFP-CF Im68, and 3.54 for NXF1 (Figure 6D). Additionally, we carried out quantitative reverse-transcription PCR (qPCR) analysis of the relative amount of luciferase mRNA in the nuclear fractions of cells transfected with different MS2-fusion constructs (Figure 6E). Quantitation of the relative nuclear luciferase RNA levels showed that tethering of MS2-CF Im68 decreased the amount of transcript in the nucleus by 2.5fold compared with MS2-REF, whereas the two CF Im68 deletion mutants (68 N and 68 C) were less effective in promoting mRNA export. The nuclear fractions were devoid of cytoplasmic contaminations as shown by Western blot analyses for lamin A/C (nuclear) and tyrosine tubulin (cytoplasmic; Supplemental Figure S4). Taken together, these experiments indicate that tethering of CF Im68 can promote mRNA export.-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	The assay described >>>promote mRNA export.
108	To further characterize this new function of CF Im68, we investigated the effect of its overexpression on the export of endogenous mRNAs. HeLa cells were transfected with a selectable plasmid driving CF Im68 expression under the CMV promoter, and the nucleo-cytoplasmic distribution of polyadenylated RNAs was examined by FISH with a Cy3labeled oligo(dT) probe (Figure 7A). In cells overexpressing CF Im68, a slight but appreciable reduction of the amount of nuclear polyadenylated RNA could be observed. To analyze the effect of CF Im68 overexpression on the nucleocytoplasmic transport of specific mRNAs, nuclear and cytoplasmic fractions were prepared, and RNA was isolated from these extracts. RT-PCR for the nuclear X-inactivation specific (X-ist) mRNA revealed that no nuclear RNA had leaked into the cytoplasmic fractions (Figure 7B). The amounts of four different mRNAs were then measured by qPCR. They are shown in Figure 7C as relative values normalized to the amounts of the same transcripts measured in cells overexpressing HA-EGFP. Importantly, the nuclear concentration of each of these transcripts was reduced two- to threefold, whereas cytoplasmic levels were increased for three of them, with the exception of ß-actin. Nevertheless, even for ß-actin mRNA, the cytoplasmic to nuclear ratio increased 2.2-fold compared with the control, indicating that CF Im68 overexpression stimulates mRNA export also in this case.-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	To further character>>>t also in this case.
109	We also determined the effect of CF Im68 depletion on mRNA export. Silencing experiments were performed in HeLa cells stably transfected with the ß-globin gene lacking natural introns and therefore expressing cDNA transcripts that do not undergo splicing (Valencia et al., 2008). The assay was validated by depleting NXF1. Two days after transfection of specific siRNAs or of a control siRNA, mRNA levels were analyzed by real-time RT-PCR. NXF1 mRNA was reduced by 92% compared with control siRNA-treated cells, and, as a consequence, the level of the nuclear ß-globin mRNA was increased 2.5 times (Supplemental Figure S5). In the case of CF Im68, 4 d after transfection of two specific siRNAs, CF Im68 mRNA was reduced by 93% compared with control siRNA-treated cells, whereas the level of the mRNA of the closely related 59-kDa subunit remained unchanged (data not shown). Western blot analysis (Figure 7D) confirmed that the level of the remaining CF Im68 protein was below detection. Importantly, this silencing of CF Im68 resulted in a small but statistically significant increase in the nuclear level of ß-globin mRNA (Figure 7E). As above, the purity of these nuclear fractions was assessed by Western blot analyses for lamin A/C and tyrosine tubulin (Supplemental Figure S4). This result supports the idea that CF Im68 may act in mRNA export, similarly to SR proteins and/or REF, by contributing to the recruitment of NXF1 to the mRNA.-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	We also determined t>>>of NXF1 to the mRNA.
110	Another variable that could potentially affect the nucleocytoplasmic distribution of mRNAs is inefficient 3 end processing that would lead to nuclear retention of the unprocessed transcript. However, CF Im68 depletion did not significantly affect the 3 end processing efficiency of neither endogenous ß-actin pre-mRNA (Figure 7F) nor of the ßglobin cDNA transcript, which has the strong polyadenylation signal of bovine growth hormone (BGH, data not shown).-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Another variable tha>>>GH, data not shown).
111	In summary, the above experiments indicate a relevant biological role for CF Im68 in the export of mRNAs.-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	In summary, the abov>>>the export of mRNAs.
112	CF I m68 Cosediments with Ribosomal Particles CBP80 and PABP2, is part of the mRNP particle that is remodeled in the cytoplasm during the initial stages of translation when nuclear proteins are replaced by their cytoplasmic counterparts (Ishigaki et al., 2001; Dostie and Dreyfuss, 2002).-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	CF I m68 Cosediments>>>and Dreyfuss, 2002).
113	Some of the components of the EJC, including REF and NXF1, dissociate from the mRNA upon entry into the cytoplasm (Le Hir et al., 2001). Therefore, it is possible that CF Im68 is present in the mRNPs in the nucleus but may then dissociate during or shortly after mRNA export. To gain insight into the remodeling of CF Im68-containing mRNPs after export, cytoplasmic fractions of HEK293 cells were fractionated across 15­50% sucrose gradients, and the distribution of CF Im68 was determined by Western blotting. Figure 8A shows that most of the cytoplasmic CF Im68 was found in lighter complexes at the top of the gradient. However, CF Im68 could also be detected in the 80S (monosome) region, although to a lesser extent than ASF/SF2. CF Im68 distribution correlated with that of the nuclear cap-binding complex component CBP80 (Figure 8B). Treatment of cytoplasmic extracts with RNaseA, which induces dissociation of mono- and polyribosomes into ribosomal subunits, led to a redistribution of CF Im68, SF2/ASF, and rpS6 to the top of the gradient (Figure 8C). Likewise, we found that cosedimentation of CF Im68 with 80S particles was sensitive to EDTA (data not shown). In contrast, treatment with puromycin, which causes premature termination and thus the disassembly of translating heavy polysomes and an increase of the 80S peak (Sabatini et al., 1971) did not affect CF Im68 distribution (Supplemental Figure S6). We conclude that CF Im68 is not associated with translating ribosomes but, like-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Some of the componen>>> ribosomes but, like
114	Nucleocytoplasmic Shuttling of CF Im-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	Nucleocytoplasmic Sh>>>c Shuttling of CF Im
115	Vol. 20, December 15, 2009-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	Vol. 20, December 15>>>0, December 15, 2009
116	5219-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	5219>>>5219
117	DISCUSSION-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	DISCUSSION>>>DISCUSSION
118	Although stimulation of mRNA export by polyadenylation has long been observed, so far little is known about the possible mechanism. In this report we provide first evidence that the 68-kDa subunit of CF Im, a pre-mRNA 3 end processing factor, interacts with the mRNA export receptor NXF1 and stimulates mRNA export. On the basis of our observations and on previous data showing that CF Im68 is associated with BrdU-labeled nascent transcripts (Cardinale et al., 2007), we propose that CF Im68 is loaded onto the pre-mRNA during cleavage and polyadenylation of the 3 end of the transcript and remains bound to the mRNA all the way to the cytoplasm where it is removed by the translation machinery. CF Im68 may thus act as a mark of correct 3 end maturation and contribute to efficient mRNA export via NXF1 recruitment.-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Although stimulation>>>ia NXF1 recruitment.
119	Role of RNA-binding Shuttling Proteins in mRNA Export-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Role of RNA-binding >>>teins in mRNA Export
120	Several RNA binding proteins exhibit shuttling activity including the splicing factor U2AF, the polyadenylation factor PABPN1, and members of the hnRNP and SR protein families (for review see Gama-Carvalho and Carmo-Fonseca, 2001). Although U2AF, PABPN1, and the hnRNP proteins-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Several RNA binding >>>d the hnRNP proteins
121	Figure 8. CF Im68 cosediments with the translation machinery. (A) HEK293 cell cytoplasmic extracts were fractionated across 15­50% sucrose gradients and analyzed by immunoblotting with ASF/SF2, CF Im68, and rpS6. (Top) UV absorbance (254 nm) profile of cytosolic ribonucleoprotein complexes. (B) Fractions were additionally analyzed by immunoblotting with ASF/SF2, CF Im68, and in addition CBP80 and PABPC1. (C) Sucrose gradient analysis of RNase A­treated cytoplasmic extracts. CF Im68, SF2/ASF, and rpS6 concentrate very narrowly across the gradient of HEK293 cytoplasmic extracts treated with RNase A, which induces dissociation of ribosomal subunits. (Top) UV absorbance (254 nm) profile of cytosolic ribonucleoprotein complexes.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->capfig	Figure 8. CF Im68 co>>>eoprotein complexes.
122	M.-D. Ruepp et al.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	M.-D. Ruepp et al.>>>M.-D. Ruepp et al.
123	Molecular Biology of the Cell-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	Molecular Biology of>>> Biology of the Cell
124	5220-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	5220>>>5220
125	were shown to shuttle actively and independently of mRNA traffic, shuttling SR proteins are exported bound to the mRNA. Similar to SR proteins, CF Im shuttling activity depends on active transcription, suggesting that it leaves the nucleus in association with the mRNA. Consistent with this observation both CF Im subunits contact the RNA substrate, as demonstrated by UV cross-linking studies (Ru¨egsegger et al., 1996).-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	were shown to shuttl>>>egger et al., 1996).
126	What is the role of shuttling proteins in mRNA export? Shuttling RNA-binding proteins may contribute to the assembly of an export competent mRNP by recruiting mRNA export factors. Consistent with this view U2AF 35-kDa subunit (Zolotukhin et al., 2002), several shuttling SR proteins (Huang et al., 2003; Lai and Tarn, 2004), and now CF Im have been shown to interact with the mRNA export receptor NXF1. In addition, shuttling RNA-binding proteins may fulfill different roles in the nucleus and in the cytoplasm. This is the case for instance for the shuttling SR protein ASF/SF2 that, besides its well-characterized role in basal and regulated splicing, is involved in mRNA surveillance and in translation (Sanford et al., 2004; Zhang and Krainer, 2004).-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	What is the role of >>> and Krainer, 2004).
127	Mechanisms for the Recruitment of the mRNA Export Receptor NXF1-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	Mechanisms for the R>>>Export Receptor NXF1
128	In S. cerevisiae where only 5% of the genes contain introns, export factor recruitment is coupled to transcription and 3 end formation rather than to intron splicing. The interaction between the mRNA and the heterodimeric export receptor Mex67/Mtr2 (NXF1/p15 in metazoa) is provided by the RNA-binding protein Yra1 (REF). Yra1 interacts with the transcription elongation complex (THO complex, Strasser et al., 2002; Abruzzi et al., 2004) and requires 3 end formation to be recruited cotranscriptionally to mRNAs (Lei and Silver, 2002). How the yeast pre-mRNA 3 end processing complex participates in the recruitment of mRNA export factors is, however, still unknown.-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	In S. cerevisiae whe>>>ever, still unknown.
129	In metazoans, the current model for the recruitment of export factors on the mRNA proposes that adaptor proteins such as REF and the export receptor NXF1 are "deposited" after splicing as components of the EJC (for review see Stutz and Izaurralde, 2003). REF is recruited by UAP56 that binds cotranscriptionally. Before export, NXF1/p15 would bind to REF and thereby release UAP56. However, only NXF1, p15, and UAP56 have been shown so far to be essential for this process (Tan et al., 2000; Herold et al., 2001; Wilkie et al., 2001; Gatfield and Izaurralde, 2002; Wiegand et al., 2002). Recently, an EJC-independent mechanism was proposed for the recruitment of REF based on the observation that UAP56 and REF bind at the 5 end of the mRNA and interact with the cap-binding complex. In this new model recruitment occurs via an interaction between REF and the cap-binding protein CBP80 (Cheng et al., 2006). The observation that depletion of REF in Drosophila cells does not lead to accumulation of poly(A)RNA in the nucleus (Gatfield and Izaurralde, 2002) suggests that alternative mechanisms must exist for the recruitment of NXF1 on mature mRNAs. Consistent with this idea, the shuttling SR proteins SRp20, 9G8, and ASF/SF2 that can bind to the pre-mRNA independently of the EJC, were shown to act as NXF1-adaptor proteins. Here, we have demonstrated that CF Im68 interacts with NXF1 and contributes to mRNA export most likely in conjunction with SR proteins. The presence of CF Im68 may signal that the mRNA has been properly cleaved and polyadenylated (Figure 9) and may be packaged into an exportcompetent mRNA ribonucleoprotein particle (mRNP) that can recruit the export receptor. This novel observation provides for the first time a molecular connection between 3 end formation and mRNA export.-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	In metazoans, the cu>>>ion and mRNA export.
130	Figure 9. Model of potential role of CF Im68 in assembly and export of mRNPs. (a) CF Im is recruited to the pre-mRNA as part of the cleavage and polyadenylation complex. (b) Once correct 3 end processing has occurred, CF Im68 remains associated with the mRNA and (c) in conjunction with SR proteins contributes to the recruitment of the export receptor NXF1. (d) In the cytoplasm, the translating ribosomes remove the nuclear proteins that are reimported into the nucleus.-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->capfig	Figure 9. Model of p>>>ed into the nucleus.
131	Nucleocytoplasmic Shuttling of CF Im-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	Nucleocytoplasmic Sh>>>c Shuttling of CF Im
132	Vol. 20, December 15, 2009-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	Vol. 20, December 15>>>0, December 15, 2009
133	5221-->id=0, page=0, size=10, fam=Times, col=#231f20, type=parrnote, textLines=182--->[]--->note	5221>>>5221
134	In contrast to NXF1, CF Im68 does not appear to dissociate from the mRNA upon entry into the cytoplasm. Instead, sucrose gradient centrifugation shows that CF Im68 cosediments with the 80S ribosome. According to current models, the first passage of the 80S ribosome on an mRNA (called "pioneer round" of translation) leads to a major rearrangement of the mRNP composition whereby the remaining nuclear mRNP components are removed and replaced by cytoplasmic factors (for review see Maquat, 2004; Moore, 2005). Thus, our findings that CFIm68 is released from the mRNP at this stage may have interesting implications not only for mRNA export and translation initiation but also for mRNA quality control mechanisms. For example, the presence of CF Im bound downstream of a stop codon may contribute to the definition of the proper termination codon in metazoa. Further studies on the role of the factors that bind the 3 end of the mRNA should provide new important insight into the mechanism that defines premature stop codons and leads to NMD.-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	In contrast to NXF1,>>>ns and leads to NMD.
135	ACKNOWLEDGMENTS-->id=8, page=0, size=11, fam=Times, col=#231f20, type=parr, textLines=711--->[]--->parr	ACKNOWLEDGMENTS>>>ACKNOWLEDGMENTS
136	We gratefully acknowledge M. Bianchi (San Raffaele University, Milan, Italy), J. Lykke-Andersen (University of Colorado, Boulder, CO), W.Y. Tarn (Academia Sinica, Taipei, Taiwan), R. Reed (Harvard Medical School, Boston, MA) and S. Wilson (University of Sheffield, Sheffield, United Kingdom) for plasmids; F. Loreni (University 'Tor Vergata,' Rome, Italy) for antibodies; E. Izuarralde (Max Planck Institute for Developmental Biology, Tu¨bingen, Germany) for plasmids and antibodies; W. Keller (University of Basel, Basel, Switzerland) for histidine-tagged CF Im subunits; and S. Stamm (University of Erlangen, Erlangen, Germany) for Sf9 cells expressing His-Tra2 . We also thank A. I. Lamond, J. Sanford, and J. Caceres for help with some of the experiments and O. Mu¨hlemann for valuable suggestions and proofreading of the manuscript. We appreciate the technical help of N. Kleinschmidt, Lukas Stalder, Sandro Waltersperger, and K. Schranz. This study was partially supported by a MIUR-PRIN 2006 grant and by the Cariplo Foundation. S.C. was supported by a short-term EMBO fellowship. M-D.R. and D.S. were supported by the Swiss National Science Foundation (Grants 3100A0-105547 and -120064) and the Canton Bern.-->id=830, page=1, size=8, fam=Times, col=#231f20, type=parrnote, textLines=407--->[]--->parr	We gratefully acknow>>>and the Canton Bern.
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