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• W2052135311 abstract "Splicing is a crucial step for human immunodeficiency virus, type 1 (HIV-1) multiplication; eight acceptor sites are used in competition to produce the vif, vpu, vpr, nef, env, tat, and rev mRNAs. The effects of SR proteins have only been investigated on a limited number of HIV-1 splicing sites by using small HIV-1 RNA pieces. To understand how SR proteins influence the use of HIV-1 splicing sites, we tested the effects of overproduction of individual SR proteins in HeLa cells on the splicing pattern of an HIV-1 RNA that contained all the splicing sites. The steady state levels of the HIV-1 mRNAs produced were quantified by reverse transcriptase-PCR. For interpretation of the data, transcripts containing one or several of the HIV-1 acceptor sites were spliced in vitro in the presence or the absence of one of the tested SR proteins. Both in vivo and in vitro, acceptor sites A2 and A3 were found to be strongly and specifically regulated by SR proteins. ASF/SF2 strongly activates site A2 and to a lesser extent site A1. As a result, upon ASF/SF2 overexpression, the vpr mRNA steady state level is specifically increased. SC35 and SRp40, but not 9G8, strongly activate site A3, and their overexpression ex vivo induces a dramatic accumulation of the tat mRNA, to the detriment of most of the other viral mRNAs. Here we showed by Western blot analysis that the Nef protein synthesis is strongly decreased by overexpression of SC35, SRp40, and ASF/SF2. Finally, activation by ASF/SF2 and 9G8 was found to be independent of the RS domain. This is the first investigation of the effects of variations of individual SR protein concentrations that is performed ex vivo on an RNA containing a complex set of splicing sites. Splicing is a crucial step for human immunodeficiency virus, type 1 (HIV-1) multiplication; eight acceptor sites are used in competition to produce the vif, vpu, vpr, nef, env, tat, and rev mRNAs. The effects of SR proteins have only been investigated on a limited number of HIV-1 splicing sites by using small HIV-1 RNA pieces. To understand how SR proteins influence the use of HIV-1 splicing sites, we tested the effects of overproduction of individual SR proteins in HeLa cells on the splicing pattern of an HIV-1 RNA that contained all the splicing sites. The steady state levels of the HIV-1 mRNAs produced were quantified by reverse transcriptase-PCR. For interpretation of the data, transcripts containing one or several of the HIV-1 acceptor sites were spliced in vitro in the presence or the absence of one of the tested SR proteins. Both in vivo and in vitro, acceptor sites A2 and A3 were found to be strongly and specifically regulated by SR proteins. ASF/SF2 strongly activates site A2 and to a lesser extent site A1. As a result, upon ASF/SF2 overexpression, the vpr mRNA steady state level is specifically increased. SC35 and SRp40, but not 9G8, strongly activate site A3, and their overexpression ex vivo induces a dramatic accumulation of the tat mRNA, to the detriment of most of the other viral mRNAs. Here we showed by Western blot analysis that the Nef protein synthesis is strongly decreased by overexpression of SC35, SRp40, and ASF/SF2. Finally, activation by ASF/SF2 and 9G8 was found to be independent of the RS domain. This is the first investigation of the effects of variations of individual SR protein concentrations that is performed ex vivo on an RNA containing a complex set of splicing sites. Splicing plays a key role for production of the HIV-1 1The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; RT, reverse transcriptase; sn, small nuclear; hnRNP, heterogeneous nuclear ribonucleoprotein; nt, nucleotide.1The abbreviations used are: HIV-1, human immunodeficiency virus, type 1; RT, reverse transcriptase; sn, small nuclear; hnRNP, heterogeneous nuclear ribonucleoprotein; nt, nucleotide. retroviral proteins. By using the integrated proviral genome as the template, RNA polymerase II of the infected cell produces long primary transcripts that are all identical. Some of these transcripts are transported to the cytoplasm in an intact form to serve as genomes for new virions or as messenger RNAs for the production of the Gag-Pol protein precursor. The other transcripts undergo alternative splicing to produce mRNAs for the auxiliary and regulatory proteins and the Env precursor protein. Production of mRNAs encoding HIV-1 proteins depends on the alternative utilization of four 5′-splice sites D1 to D4 (1Purcell D.F. Martin M.A. J. Virol. 1993; 67: 6365-6378Crossref PubMed Google Scholar) and eight 3′-splice sites (A1, A2, A3, A4a, -b, -c, A5, and A7) (1Purcell D.F. Martin M.A. J. Virol. 1993; 67: 6365-6378Crossref PubMed Google Scholar). An additional 3′-splice site (A6) was only found in one HIV-1 strain (2Benko D.M. Schwartz S. Pavlakis G.N. Felber B.K. J. Virol. 1990; 64: 2505-2518Crossref PubMed Google Scholar). Donor sites D1, D2, and D3 can be coupled to any of the A1 to A5 acceptor sites, whereas donor site D4 is exclusively coupled to site A7 and to site A6 when this site is present (1Purcell D.F. Martin M.A. J. Virol. 1993; 67: 6365-6378Crossref PubMed Google Scholar, 2Benko D.M. Schwartz S. Pavlakis G.N. Felber B.K. J. Virol. 1990; 64: 2505-2518Crossref PubMed Google Scholar). The combination of these various sites gives rise to at least 35 different mRNAs (1Purcell D.F. Martin M.A. J. Virol. 1993; 67: 6365-6378Crossref PubMed Google Scholar). Although the relative efficiencies of the HIV-1 donor sites seem to depend mainly upon their complementarity to the U1 snRNA 5′-terminal sequence (3O'Reilly M.M. McNally M.T. Beemon K.L. Virology. 1995; 213: 373-385Crossref PubMed Scopus (101) Google Scholar, 4, Damier, L. (1997) Etude de la Régulation de l'Épissage du Transcrit Primaire du Virus de l'Immunodéficience Humaine de Type 1, HIV-1. Ph.D. thesis, Université Henri Poincaré, Nancy, FranceGoogle Scholar), efficiencies of HIV-1 acceptor sites depend upon the presence of cis-regulatory elements (5Staffa A. Cochrane A. J. Virol. 1994; 68: 3071-3079Crossref PubMed Google Scholar, 6Staffa A. Cochrane A. Mol. Cell. Biol. 1995; 15: 4597-4605Crossref PubMed Scopus (152) Google Scholar, 7Amendt B.A. Hesslein D. Chang L.J. Stoltzfus C.M. Mol. Cell. Biol. 1994; 14: 3960-3970Crossref PubMed Scopus (125) Google Scholar, 8Amendt B.A. Si Z.H. Stoltzfus C.M. Mol. Cell. Biol. 1995; 15: 4606-4615Crossref PubMed Scopus (123) Google Scholar, 9Si Z. Amendt B.A. Stoltzfus C.M. Nucleic Acids Res. 1997; 25: 861-867Crossref PubMed Scopus (86) Google Scholar, 10Si Z.H. Rauch D. Stoltzfus C.M. Mol. Cell. Biol. 1998; 18: 5404-5413Crossref PubMed Scopus (68) Google Scholar, 11Jacquenet S. Mereau A. Bilodeau P.S. Damier L. Stoltzfus C.M. Branlant C. J. Biol. Chem. 2001; 276: 40464-40475Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 12Bilodeau P.S. Domsic J.K. Mayeda A. Krainer A.R. Stoltzfus C.M. J. Virol. 2001; 75: 8487-8497Crossref PubMed Scopus (99) Google Scholar, 13Bilodeau P.S. Domsic J.K. Stoltzfus C.M. J. Virol. 1999; 73: 9764-9772Crossref PubMed Google Scholar, 14Caputi M. Mayeda A. Krainer A.R. Zahler A.M. EMBO J. 1999; 18: 4060-4067Crossref PubMed Scopus (225) Google Scholar, 15Caputi M. Zahler A.M. EMBO J. 2002; 21: 845-855Crossref PubMed Scopus (103) Google Scholar, 16Del Gatto-Konczak F. Olive M. Gesnel M.C. Breathnach R. Mol. Cell. Biol. 1999; 19: 251-260Crossref PubMed Scopus (198) Google Scholar, 17Tange T.O. Kjems J. J. Mol. Biol. 2001; 312: 649-662Crossref PubMed Scopus (50) Google Scholar, 18Tange T.O. Damgaard C.K. Guth S. Valcarcel J. Kjems J. EMBO J. 2001; 20: 5748-5758Crossref PubMed Scopus (133) Google Scholar, 19Zhu J. Mayeda A. Krainer A.R. Mol. Cell. 2001; 8: 1351-1361Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 20Pongoski J. Asai K. Cochrane A. J. Virol. 2002; 76: 5108-5120Crossref PubMed Scopus (35) Google Scholar, 21Marchand V. Mereau A. Jacquenet S. Thomas D. Mougin A. Gattoni R. Stevenin J. Branlant C. J. Mol. Biol. 2002; 323: 629-652Crossref PubMed Scopus (83) Google Scholar). Several studies have shown that HIV-1 acceptor sites are suboptimal as follows. (i) Their polypyrimidine tracts are short and interrupted by purines (3O'Reilly M.M. McNally M.T. Beemon K.L. Virology. 1995; 213: 373-385Crossref PubMed Scopus (101) Google Scholar, 5Staffa A. Cochrane A. J. Virol. 1994; 68: 3071-3079Crossref PubMed Google Scholar, 9Si Z. Amendt B.A. Stoltzfus C.M. Nucleic Acids Res. 1997; 25: 861-867Crossref PubMed Scopus (86) Google Scholar). (ii) Their branch point sequences are not canonical, and in some cases, a residue other than an adenosine is used as the branch site (22Dyhr-Mikkelsen H. Kjems J. J. Biol. Chem. 1995; 270: 24060-24066Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 23Damier L. Domenjoud L. Branlant C. Biochem. Biophys. Res. Commun. 1997; 237: 182-187Crossref PubMed Scopus (22) Google Scholar). (iii) Their accessibility is limited by their sequestering in stable secondary structures (24Jacquenet S. Ropers D. Bilodeau P.S. Damier L. Mougin A. Stoltzfus C.M. Branlant C. Nucleic Acids Res. 2001; 29: 464-478Crossref PubMed Scopus (66) Google Scholar). (iv) Several cis-inhibitory elements have been identified that down-regulate the A2, A3, and A7 sites by binding of hnRNP A1 or hnRNP H proteins (11Jacquenet S. Mereau A. Bilodeau P.S. Damier L. Stoltzfus C.M. Branlant C. J. Biol. Chem. 2001; 276: 40464-40475Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 12Bilodeau P.S. Domsic J.K. Mayeda A. Krainer A.R. Stoltzfus C.M. J. Virol. 2001; 75: 8487-8497Crossref PubMed Scopus (99) Google Scholar, 14Caputi M. Mayeda A. Krainer A.R. Zahler A.M. EMBO J. 1999; 18: 4060-4067Crossref PubMed Scopus (225) Google Scholar, 16Del Gatto-Konczak F. Olive M. Gesnel M.C. Breathnach R. Mol. Cell. Biol. 1999; 19: 251-260Crossref PubMed Scopus (198) Google Scholar, 18Tange T.O. Damgaard C.K. Guth S. Valcarcel J. Kjems J. EMBO J. 2001; 20: 5748-5758Crossref PubMed Scopus (133) Google Scholar, 19Zhu J. Mayeda A. Krainer A.R. Mol. Cell. 2001; 8: 1351-1361Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 21Marchand V. Mereau A. Jacquenet S. Thomas D. Mougin A. Gattoni R. Stevenin J. Branlant C. J. Mol. Biol. 2002; 323: 629-652Crossref PubMed Scopus (83) Google Scholar). Despite these numerous handicaps, the utilization of several of the HIV-1 acceptor sites is essential for virus multiplication. Sites A3 and A7 are used for production of mRNAs of the Tat transcriptional activator (see Ref. 1Purcell D.F. Martin M.A. J. Virol. 1993; 67: 6365-6378Crossref PubMed Google Scholar and for review see Ref. 25Karn J. J. Mol. Biol. 1999; 293: 235-254Crossref PubMed Scopus (388) Google Scholar). One of the three A4a, -b, or -c sites and site A7 are used to produce mRNAs for the Rev protein (1Purcell D.F. Martin M.A. J. Virol. 1993; 67: 6365-6378Crossref PubMed Google Scholar). This regulatory protein plays an essential role in the transport of intact primary transcripts to the cytoplasm (for review see Ref. 26Boris-Lawrie K. Roberts T.M. Hull S. Life Sci. 2001; 69: 2697-2709Crossref PubMed Scopus (30) Google Scholar). Site A5 is required for production of mRNAs for the Nef and Vpu proteins. Nef down-regulates the cell-surface expression of the HIV-1 receptor glycoprotein and has many additional roles in the infected cell that favor virus multiplication. It is also responsible for AIDS pathology, because of its role in bystander lymphocyte apoptosis (for reviews see Refs. 27Harris M. Curr. Biol. 1999; 9: R459-R461Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar and 28Azad A.A. Biochem. Biophys. Res. Commun. 2000; 267: 677-685Crossref PubMed Scopus (55) Google Scholar). Finally, site A2 is needed for production of mRNAs for the Vpr protein. Vpr is implicated in nuclear import of the HIV-1 pre-integration complex, and it induces cell cycle arrest in proliferating cells (for review see Ref. 29Bukrinsky M. Adzhubei A. Rev. Med. Virol. 1999; 9: 39-49Crossref PubMed Scopus (78) Google Scholar). The efficiency of the regulated acceptor sites of cellular mRNAs generally depends upon the binding of members of the SR protein family (for reviews see Refs. 30Graveley B.R. RNA (N. Y.). 2000; 6: 1197-1211Crossref PubMed Scopus (878) Google Scholar and 31Smith C.W. Valcarcel J. Trends Biochem. Sci. 2000; 25: 381-388Abstract Full Text Full Text PDF PubMed Scopus (752) Google Scholar). To date, 10 distinct members of this family have been identified (for reviews see Refs. 30Graveley B.R. RNA (N. Y.). 2000; 6: 1197-1211Crossref PubMed Scopus (878) Google Scholar and 32Manley J.L. Tacke R. Genes Dev. 1996; 10: 1569-1579Crossref PubMed Scopus (601) Google Scholar). These proteins contain one or two RNA recognition motif(s), together with a carboxyl-terminal domain containing numerous arginine-serine amino acid pairs. SR proteins are involved in constitutive splicing where they seem to have redundant functions in assistance to spliceosomal component assembly. Because of their interactions with the 70K protein of U1 snRNP, the 35-kDa subunit of factor U2AF, and proteins of the U4/U6.U5 tri-snRNP, the SR proteins are essential actors in the complex mechanism of spliceosome assembly (for reviews see Refs. 30Graveley B.R. RNA (N. Y.). 2000; 6: 1197-1211Crossref PubMed Scopus (878) Google Scholar and 33Hastings M.L. Krainer A.R. Curr. Opin. Cell Biol. 2001; 13: 302-309Crossref PubMed Scopus (386) Google Scholar). In addition, SR proteins can regulate alternative splicing by binding to cis-regulatory elements. These regulatory elements generally bind specifically one given SR protein and, depending on the identity of this SR protein, different effects may be generated. Binding of SR proteins to specific sequences located downstream from the acceptor site generally reinforces the efficiency of U2AF binding to the polypyrimidine tract, either by direct interaction of the RS domain of the SR protein with the RS domain of U2AF35 or by displacement of the hnRNP A1 protein that blocks the access of spliceosomal components to the 3′-splice site (for reviews see Refs. 30Graveley B.R. RNA (N. Y.). 2000; 6: 1197-1211Crossref PubMed Scopus (878) Google Scholar, 31Smith C.W. Valcarcel J. Trends Biochem. Sci. 2000; 25: 381-388Abstract Full Text Full Text PDF PubMed Scopus (752) Google Scholar, and 33Hastings M.L. Krainer A.R. Curr. Opin. Cell Biol. 2001; 13: 302-309Crossref PubMed Scopus (386) Google Scholar). The role of SR proteins on viral multiplication has been demonstrated by previous studies on adenovirus. SR proteins activate the production of the early mRNAs, but a limitation of their activity is required for the late phase of infection (34Molin M. Akusjarvi G. J. Virol. 2000; 74: 9002-9009Crossref PubMed Scopus (31) Google Scholar). This limitation results from titration of the SR proteins by the major late transcripts (35Himmelspach M. Cavaloc Y. Chebli K. Stevenin J. Gattoni R. RNA (N. Y.). 1995; 1: 794-806PubMed Google Scholar) and/or a virus-induced dephosphorylation (36Kanopka A. Muhlemann O. Petersen-Mahrt S. Estmer C. Ohrmalm C. Akusjarvi G. Nature. 1998; 393: 185-187Crossref PubMed Scopus (166) Google Scholar). Although variations of the cellular concentrations of some of the SR proteins, namely SC35 and 9G8, have been observed after cell infection by virus HIV-1 (37Maldarelli F. Xiang C. Chamoun G. Zeichner S.L. Virus Res. 1998; 53: 39-51Crossref PubMed Scopus (29) Google Scholar, 38Ryo A. Suzuki Y. Arai M. Kondoh N. Wakatsuki T. Hada A. Shuda M. Tanaka K. Sato C. Yamamoto M. Yamamoto N. AIDS Res. Hum. Retroviruses. 2000; 16: 995-1005Crossref PubMed Scopus (38) Google Scholar), little is known about the action of SR proteins on the competition between the eight splicing acceptor sites and four splicing donor sites of HIV-1 RNA. The present knowledge in this field can be summarized as follows: (i) deep studies, which were performed both in vitro and in vivo, demonstrated the action of proteins ASF/SF2 and SC35 at acceptor site A7 (17Tange T.O. Kjems J. J. Mol. Biol. 2001; 312: 649-662Crossref PubMed Scopus (50) Google Scholar, 19Zhu J. Mayeda A. Krainer A.R. Mol. Cell. 2001; 8: 1351-1361Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 20Pongoski J. Asai K. Cochrane A. J. Virol. 2002; 76: 5108-5120Crossref PubMed Scopus (35) Google Scholar, 39Fu X.D. Mayeda A. Maniatis T. Krainer A.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11224-11228Crossref PubMed Scopus (198) Google Scholar, 40Mayeda A. Screaton G.R. Chandler S.D. Fu X.D. Krainer A.R. Mol. Cell. Biol. 1999; 19: 1853-1863Crossref PubMed Scopus (128) Google Scholar); (ii) an in vitro study performed with S100 extract showed that, among the SR proteins SC35, SRp40, SRp55, and SRp70, only protein SC35 is able to activate site A3 in S100 extract (41Zahler A.M. Damgaard C.K. Kjems J. Caputi M. J. Biol. Chem. 2004; 279: 10077-10084Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). For a complete study of the effects of individual SR proteins on the entire set of HIV-1 splicing sites in cellular conditions, we cotransfected HeLa cells with a construct that produces a truncated HIV-1 RNA containing all the splicing sites (13Bilodeau P.S. Domsic J.K. Stoltzfus C.M. J. Virol. 1999; 73: 9764-9772Crossref PubMed Google Scholar, 24Jacquenet S. Ropers D. Bilodeau P.S. Damier L. Mougin A. Stoltzfus C.M. Branlant C. Nucleic Acids Res. 2001; 29: 464-478Crossref PubMed Scopus (66) Google Scholar), and a plasmid that overexpresses one of the SC35, SRp40, ASF/SF2, and 9G8 SR proteins. In these ex vivo experiments, very different HIV-1 RNA splicing patterns were obtained with different overexpressed SR protein. For interpretation of the data, in vitro splicing assays were performed with transcripts that contained the D1 donor site and one or several of the A1 to A5 acceptor sites. Plasmids Used in This Study—pLD-C2, pLD-C3, and pLD-L3-U1 constructs, used for production of the C2, C3, and L3-U1 transcripts, were described previously (11Jacquenet S. Mereau A. Bilodeau P.S. Damier L. Stoltzfus C.M. Branlant C. J. Biol. Chem. 2001; 276: 40464-40475Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 23Damier L. Domenjoud L. Branlant C. Biochem. Biophys. Res. Commun. 1997; 237: 182-187Crossref PubMed Scopus (22) Google Scholar, 24Jacquenet S. Ropers D. Bilodeau P.S. Damier L. Mougin A. Stoltzfus C.M. Branlant C. Nucleic Acids Res. 2001; 29: 464-478Crossref PubMed Scopus (66) Google Scholar). To build the plasmid pLD-C1, two DNA fragments were generated by PCR amplifications of plasmid pBRU3 (42Charneau P. Alizon M. Clavel F. J. Virol. 1992; 66: 2814-2820Crossref PubMed Google Scholar) containing the HIV-1 BRU/LAI complete cDNA (Gen-Bank™ accession number K02013). Amplifications were performed as described previously (23Damier L. Domenjoud L. Branlant C. Biochem. Biophys. Res. Commun. 1997; 237: 182-187Crossref PubMed Scopus (22) Google Scholar). The DNA fragment encoding the RNA region from position 4313 to 4545 (43Ratner L. Haseltine W. Patarca R. Livak K.J. Starcich B. Josephs S.F. Doran E.R. Rafalski J.A. Whitehorn E.A. Baumeister K. Ivanoff L. Petteway Jr., S.R. Pearson M.L. Lautenberger J.A. Papas T.S. Ghrayeb J. Chang N.T. Gallo R.C. Wong-Staal F. Nature. 1985; 313: 277-284Crossref PubMed Scopus (1727) Google Scholar) was amplified with the following: (i) a sense primer O-756 (5′-TATTCTAGATCTCAGGCTGAACATC-3′) containing the HIV-1 BRU RNA sequence from positions 4313 to 4325 (underlined) and a BglII restriction site (italic); and (ii) an antisense primer O-764 (5′-CGGAATTCCTTTCCAGAGGAGCT-3′) complementary to the HIV-1 BRU RNA from positions 4530 to 4545 (underlined) and containing an EcoRI restriction site (italic). The BglII-EcoRI fragment of plasmid pLD-C2 (23Damier L. Domenjoud L. Branlant C. Biochem. Biophys. Res. Commun. 1997; 237: 182-187Crossref PubMed Scopus (22) Google Scholar) was substituted for the BglII-EcoRI fragment of the amplified DNA. The SP1 ex2 inv 9G8–102 plasmid (44Cavaloc Y. Bourgeois C.F. Kister L. Stevenin J. RNA (N. Y.). 1999; 5: 468-483Crossref PubMed Scopus (179) Google Scholar) was used for production of an SP6 transcript allowing us to test for protein 9G8 splicing activation properties in vitro. Plasmid ΔPSP used for transfection experiments was described previously (11Jacquenet S. Mereau A. Bilodeau P.S. Damier L. Stoltzfus C.M. Branlant C. J. Biol. Chem. 2001; 276: 40464-40475Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Plasmids pXJ41-ASF, pXJ41-SRp40, pXJ42–9G8, pXJ42-SC35, or pXJ42-SRp20 were used for overexpression of the corresponding SR proteins. They were constructed by insertion of the cDNA of each SR protein in the multicloning site of pXJ41 or pXJ42 expression vector, as described previously (45Bourgeois C.F. Popielarz M. Hildwein G. Stevenin J. Mol. Cell. Biol. 1999; 19: 7347-7356Crossref PubMed Scopus (61) Google Scholar). In Vitro Transcription and in Vitro Splicing Assays—Plasmids pLD-C1 and pLD-C2 were linearized by EcoRI, plasmids pLD-C3 and pLD-L3-U1 by PstI, and plasmid Sp1 ex2 inv 9G8-102 by HindIII. They were used as templates for the production of capped, uniformly labeled RNAs by the T7 or SP6 RNA polymerase in the presence of [α-32P]UTP. In vitro splicing assays were performed in HeLa cell nuclear extract (from the Computer Cell Culture Center S.A., Belgium), pure cytoplasmic S100 extract prepared as described previously (35Himmelspach M. Cavaloc Y. Chebli K. Stevenin J. Gattoni R. RNA (N. Y.). 1995; 1: 794-806PubMed Google Scholar), or mixtures of S100 and nuclear extracts (4:4 μl and 7:1 μl), in 22-μl assays using classical conditions for in vitro splicing reaction (23Damier L. Domenjoud L. Branlant C. Biochem. Biophys. Res. Commun. 1997; 237: 182-187Crossref PubMed Scopus (22) Google Scholar). The duration of incubation at 30 °C was for 2 h and 30 min. For SR protein assays, 150 or 300 ng of purified 9G8, ASF/SF2, SC35, or SRp40 recombinant protein were used, prepared as described previously (44Cavaloc Y. Bourgeois C.F. Kister L. Stevenin J. RNA (N. Y.). 1999; 5: 468-483Crossref PubMed Scopus (179) Google Scholar). The degree of purity of these proteins was verified by electrophoresis followed by argentic staining and Western blot analysis. The C1, C2, C3, L3-U1, and Sp1 ex2 inv 9G8-102 splicing products were analyzed by electrophoresis on 5 or 6% polyacrylamide denaturing gels and were visualized by autoradiography. Quantifications were performed with a Phosphor-Imager (Amersham Biosciences) using the ImageQuant software (version 5.2). Splicing efficiency was expressed as the ratio of spliced products (M) versus unspliced RNA (P). For each gel electrophoresis of splicing products, M/P values were measured three times using the ImageQuant software. The mean value is given with an estimation of the M/P measurement error, shown as bar errors in the histograms representing the M/P values (Figs.1, E1–E4, 2, C1–C4, and 3, C1–C3). The factor of splicing activation by SR proteins was estimated by dividing the M/P ratio obtained in the presence of the recombinant SR protein by that obtained in its absence (fold of activation). The estimated errors on the factors of activation due to the experimental errors on M/P measurements mentioned above was deduced and represented as bar errors in the histograms showing activation factors.Fig. 2Differential effects of individual SR proteins on the splicing of the L3-U1 RNA in S100 or NE/S100 mixtures. A, schematic representation of the L3-U1 pre-mRNA containing the donor site D1 and the acceptor sites A3 to A5. The representation is as shown in Fig. 1. The various splicing sites and splicing regulatory elements that were previously identified are also represented (7Amendt B.A. Hesslein D. Chang L.J. Stoltzfus C.M. Mol. Cell. Biol. 1994; 14: 3960-3970Crossref PubMed Scopus (125) Google Scholar, 8Amendt B.A. Si Z.H. Stoltzfus C.M. Mol. Cell. Biol. 1995; 15: 4606-4615Crossref PubMed Scopus (123) Google Scholar, 9Si Z. Amendt B.A. Stoltzfus C.M. Nucleic Acids Res. 1997; 25: 861-867Crossref PubMed Scopus (86) Google Scholar, 11Jacquenet S. Mereau A. Bilodeau P.S. Damier L. Stoltzfus C.M. Branlant C. J. Biol. Chem. 2001; 276: 40464-40475Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 14Caputi M. Mayeda A. Krainer A.R. Zahler A.M. EMBO J. 1999; 18: 4060-4067Crossref PubMed Scopus (225) Google Scholar, 16Del Gatto-Konczak F. Olive M. Gesnel M.C. Breathnach R. Mol. Cell. Biol. 1999; 19: 251-260Crossref PubMed Scopus (198) Google Scholar, 41Zahler A.M. Damgaard C.K. Kjems J. Caputi M. J. Biol. Chem. 2004; 279: 10077-10084Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). B, in vitro splicing assays with uniformly labeled L3-U1 RNA were carried out as indicated above the lanes, and the splicing products were fractionated as described in Fig. 1. Positions of the precursor (P), first exon (E), and the various maturation products spliced at sites A3 (MA3), A4c (MA4c), A4a, A4b (MA4a, -b) or A5 (MA5) are indicated on the left side of the panel. Lane M corresponds to a DNA size marker. The size of the fragments in base pairs are indicated on the right side of the panel. C1 and C2, histograms representing the splicing efficiencies at site A3 in S100 extract (C1) and sites A3, A4a, A4b, and A5 in the 4:4 NE/S100 mixture, with or without addition of recombinant SR protein (C2). These efficiencies were estimated as in Fig. 1. C3 and C4, histograms showing the factor of activation by SR proteins in the 4:4 (C3) or the 1:7 (C4) NE/S100 mixture. The factors of activation in C3 were calculated from data shown in B, and the factors of activation in C4 were calculated from data that are not shown.View Large Image Figure ViewerDownload (PPT)Fig. 3Differential effects of individual SR proteins on the in vitro splicing efficiency of the L3-U1 RNA in nuclear extract. A, in vitro splicing assays with uniformly labeled L3-U1 RNA. Assays were performed in 8 μl of nuclear extract in the absence (lane 2) or the presence of the SR proteins 9G8, ASF/SF2, SC35, or SRp40 (lanes 3–6, respectively). The presence of one or more unidentified lariat compounds formed in the presence of nuclear extract (NE) is indicated. B, effect of SR protein concentration on splicing efficiency. Transcript L3-U1 was spliced in a nuclear extract in the presence of 150 or 300 ng of the SR proteins SC35 or SRp40. A and B, the positions of the lariat compounds (L), precursor RNAs (P), and maturation products (MA3, MA4a, b, and MA5) are shown on the left side of the panel. Lane M is a DNA size marker. C1, histogram showing the M/P values at sites A3 and A5 in the experiment shown in A. Error bars represent the estimated errors of M/P values, linked to the measurement of the radioactivity in the bands of the gel. C2 and C3, factors of activation by SR proteins observed in the experiments shown in A and B, respectively.View Large Image Figure ViewerDownload (PPT) Ex Vivo Splicing Assays—HeLa cells in 60-mm plastic Petri dishes were cotransfected by the calcium phosphate coprecipitation technique as described previously (46Chen C. Okayama H. Mol. Cell. Biol. 1987; 7: 2745-2752Crossref PubMed Scopus (4816) Google Scholar), with 1 μgof ΔPSP plasmid and 50–400 ng of plasmids overexpressing SR proteins. Carrier BSSK DNA was added to the transfections up to a total amount of DNA of 8 μg. Total cellular RNA was isolated from the transfected HeLa cells 48 h after transfection with the RNA-solv Reagent (Omega Bio-Tek), and it was treated by DNase. Three μg of RNA were reverse-transcribed by the Moloney murine leukemia virus-reverse transcriptase according to the supplier's specifications (Invitrogen), using oligonucleotide ACC15 (5′-TTCACTAATCGAATGGATC-3′, complementary to the HIV-1 pNL4-3 RNA from positions 8445 to 8463) as the primer. About 10% of each of the RT assays was PCR-amplified with the forward oligonucleotide 2143 (5′-GGCTTGCTGAAGCGCGCACGGCAAGAGG-3′, containing the HIV-1 pNL4–3 RNA sequence from positions 700–727), and reverse primer 2144, which spans the D4 and A7 splice sites (5′-TTGGAGGTGGGTTGCTTTGATAGAG-3′, complementary to pNL4–3 RNA from positions 6029–6041 and 8369–8381). This selectively amplified the 1.8-kb HIV size class mRNAs. Amplification was limited to 30 cycles, in order to detect both major and minor bands. DNA products were analyzed by electrophoresis on a 6% polyacrylamide nondenaturing gel, and a Typhoon 8600 imager (Amersham Biosciences) was used to scan the gel after ethidium bromide staining. PCR products were quantified using ImageQuant (Amersham Biosciences). The fractionated cDNAs were identified by reference to previous data on HeLa cells transfected with plasmid ΔPSP (11Jacquenet S. Mereau A. Bilodeau P.S. Damier L. Stoltzfus C.M. Branlant C. J. Biol. Chem. 2001; 276: 40464-40475Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 24Jacquenet S. Ropers D. Bilodeau P.S. Damier L. Mougin A. Stoltzfus C.M. Branlant C. Nucleic Acids Res. 2001; 29: 464-478Crossref PubMed Scopus (66) Google Scholar). The level of expression of individual SR proteins in the different transfection experiments was estimated by Western blot analysis of total cell extracts using the monoclonal antibody 10H3 directed against the RS domain of the SR protein family and specific antibodies directed against ASF/SF2, SC35, or 9G8 (44Cavaloc Y. Bourgeois C.F. Kister L. Stevenin J. RNA (N. Y.). 1999; 5: 468-483Crossref PubMed Scopus (179) Google Scholar). A monoclonal antibody directed against the HIV-1 Nef protein (MaTG0020), a generous gift of A. M. Aubertin, was used to test by W" @default.
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• W2052135311 title "Differential Effects of the SR Proteins 9G8, SC35, ASF/SF2, and SRp40 on the Utilization of the A1 to A5 Splicing Sites of HIV-1 RNA" @default.
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