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The Shuttling SR Protein 9G8 Plays a Role in Translation of Unspliced mRNA Containing a Constitutive Transport Element

2007; Elsevier BV; Volume: 282; Issue: 27 Linguagem: Inglês

10.1074/jbc.m701660200

ISSN

1083-351X

Autores

Jennifer E. Swartz, Yeou-Cherng Bor, Yukiko Misawa, David Rekosh, Marie‐Louise Hammarskjöld,

Tópico(s)

Viral Infections and Immunology Research

Resumo

The splicing regulatory SR protein, 9G8, has recently been proposed to function in mRNA export in conjunction with the export protein, Tap/NXF1. Tap interacts directly with the Mason-Pfizer monkey virus constitutive transport element (CTE), an element that enables export of unspliced, intron-containing mRNA. Based on our previous finding that Tap can promote polysome association and translation of CTE-RNA, we investigated the effect of 9G8 on cytoplasmic RNA fate. 9G8 was shown to enhance expression of unspliced RNA containing either the Mason-Pfizer monkey virus-CTE or the recently discovered Tap-CTE. 9G8 also enhanced polyribosome association of unspliced RNA containing a CTE. Hyperphosphorylated 9G8 was present in monosomes and small polyribosomes, whereas soluble fractions contained only hypophosphorylated protein. Our results are consistent with a model in which hypophosphorylated SR proteins remain stably associated with messenger ribonucleoprotein (mRNP) complexes during export and are released during translation initiation concomitant with increased phosphorylation. These results provide further evidence for crucial links between RNA splicing, export and translation. The splicing regulatory SR protein, 9G8, has recently been proposed to function in mRNA export in conjunction with the export protein, Tap/NXF1. Tap interacts directly with the Mason-Pfizer monkey virus constitutive transport element (CTE), an element that enables export of unspliced, intron-containing mRNA. Based on our previous finding that Tap can promote polysome association and translation of CTE-RNA, we investigated the effect of 9G8 on cytoplasmic RNA fate. 9G8 was shown to enhance expression of unspliced RNA containing either the Mason-Pfizer monkey virus-CTE or the recently discovered Tap-CTE. 9G8 also enhanced polyribosome association of unspliced RNA containing a CTE. Hyperphosphorylated 9G8 was present in monosomes and small polyribosomes, whereas soluble fractions contained only hypophosphorylated protein. Our results are consistent with a model in which hypophosphorylated SR proteins remain stably associated with messenger ribonucleoprotein (mRNP) complexes during export and are released during translation initiation concomitant with increased phosphorylation. These results provide further evidence for crucial links between RNA splicing, export and translation. The serine/arginine (SR) 3The abbreviations used are: SR protein, serine/arginine-rich protein; RS domain, arginine/serine-rich domain; CTE, constitutive transport element; MPMV, Mason-Pfizer monkey virus; HIV, human immunodeficiency virus; RRE, Rev-responsive element; SEAP, secreted alkaline phosphatase; mAb104, monoclonal antibody 104; ZnK, zinc knuckle domain; ECL, enhanced chemiluminescent assay; RSB, resuspension buffer; 9G8ΔRS, 9G8 without the RS domain; CIAP, calf intestinal alkaline phosphatase; ivt, in vitro transcribed; RNP, ribonucleoprotein; mRNP, messenger RNP; hnRNP, heterogeneous nuclear RNP. proteins are a family of RNA binding proteins with well recognized roles in splicing regulation (1Fu X.D. RNA (N. Y.). 1995; 1: 663-680PubMed Google Scholar, 2Graveley B.R. RNA (N. Y.). 2000; 6: 1197-1211Crossref PubMed Scopus (884) Google Scholar, 3Valcarcel J. Green M.R. Trends Biochem. Sci. 1996; 21: 296-301Abstract Full Text PDF PubMed Google Scholar). These proteins all have C-terminal arginineand serinerich (RS) domains that can be phosphorylated and dephosphorylated on multiple serines by cellular kinases and phosphatases (2Graveley B.R. RNA (N. Y.). 2000; 6: 1197-1211Crossref PubMed Scopus (884) Google Scholar). The phosphorylation state is believed to play an important role in functional regulation (2Graveley B.R. RNA (N. Y.). 2000; 6: 1197-1211Crossref PubMed Scopus (884) Google Scholar, 4Cao W. Jamison S.F. Garcia-Blanco M.A. RNA (N. Y.). 1997; 3: 1456-1467PubMed Google Scholar, 5Mermoud J.E. Cohen P.T. Lamond A.I. EMBO J. 1994; 13: 5679-5688Crossref PubMed Scopus (276) Google Scholar, 6Yun C.Y. Velazquez-Dones A.L. Lyman S.K. Fu X.D. J. Biol. Chem. 2003; 278: 18050-18055Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The effects of SR proteins on splicing have been extensively studied. Based to a large extent on in vitro studies, it has been well established that SR proteins, in general, bind to cis-acting regulatory sequences in many RNAs and promote splicing (7Sanford J.R. Longman D. Caceres J.F. Prog. Mol. Subcell Biol. 2003; 31: 33-58Crossref PubMed Scopus (60) Google Scholar, 8Tacke R. Manley J.L. Curr. Opin. Cell Biol. 1999; 11: 358-362Crossref PubMed Scopus (181) Google Scholar). Although individual proteins can have specific effects, SR proteins often appear to display overlapping and redundant functions (2Graveley B.R. RNA (N. Y.). 2000; 6: 1197-1211Crossref PubMed Scopus (884) Google Scholar, 8Tacke R. Manley J.L. Curr. Opin. Cell Biol. 1999; 11: 358-362Crossref PubMed Scopus (181) Google Scholar). The SR proteins were initially thought to have solely nuclear functions, but it was subsequently shown that several of these proteins shuttle between the nucleus and the cytoplasm (9Caceres J.F. Screaton G.R. Krainer A.R. Genes Dev. 1998; 12: 55-66Crossref PubMed Scopus (394) Google Scholar). The functional importance of this remained unclear until studies by Steitz and colleagues demonstrated a potential role for two of the shuttling SR proteins (SRp20 and 9G8) in mRNA export (10Huang Y. Steitz J.A. Mol. Cell. 2001; 7: 899-905Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 11Huang Y. Gattoni R. Stevenin J. Steitz J.A. Mol. Cell. 2003; 11: 837-843Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 12Huang Y. Yario T.A. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9666-9670Crossref PubMed Scopus (197) Google Scholar). Another study reported the presence of SR proteins in polyribosomes, suggesting a potential role in translational regulation (13Sanford J.R. Gray N.K. Beckmann K. Caceres J.F. Genes Dev. 2004; 18: 755-768Crossref PubMed Scopus (303) Google Scholar). Specifically, it was reported that the SF2/ASF protein was able to promote translation of mRNAs from reporter constructs containing SR protein binding sites. Several recent studies have now provided additional support of a role for SR proteins in translation (14Blaustein M. Pelisch F. Tanos T. Munoz M.J. Wengier D. Quadrana L. Sanford J.R. Muschietti J.P. Kornblihtt A.R. Caceres J.F. Coso O.A. Srebrow A. Nat. Struct. Mol. Biol. 2005; 12: 1037-1044Crossref PubMed Scopus (192) Google Scholar, 15Sanford J.R. Ellis J.D. Cazalla D. Caceres J.F. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15042-15047Crossref PubMed Scopus (103) Google Scholar). In the case of most mammalian genes, the primary RNA transcript contains multiple introns that must be removed by splicing before the mRNA can exit the nucleus (16Cullen B.R. J. Cell Sci. 2003; 116: 587-597Crossref PubMed Scopus (179) Google Scholar, 17Dreyfuss G. Kim V.N. Kataoka N. Nat. Rev. Mol. Cell Biol. 2002; 3: 195-205Crossref PubMed Scopus (1132) Google Scholar, 18Luo M.J. Reed R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14937-14942Crossref PubMed Scopus (304) Google Scholar). Although it is not clear what restricts mRNA from export before splicing has been completed, the shuttling protein, Tap/NXF1 (hereafter referred to as Tap), is believed to play an important role in the export process of many mRNAs. It has been suggested that Tap is recruited to completely spliced mRNAs through multiple adaptor proteins (19Stutz F. Izaurralde E. Trends Cell Biol. 2003; 13: 319-327Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 20Reed R. Hurt E. Cell. 2002; 108: 523-531Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). In addition to the initially identified adaptor protein REF (21Luo M.L. Zhou Z. Magni K. Christoforides C. Rappsilber J. Mann M. Reed R. Nature. 2001; 413: 644-647Crossref PubMed Scopus (308) Google Scholar, 22Strasser K. Masuda S. Mason P. Pfannstiel J. Oppizzi M. Rodriguez-Navarro S. Rondon A.G. Aguilera A. Struhl K. Reed R. Hurt E. Nature. 2002; 417: 304-308Crossref PubMed Scopus (645) Google Scholar), three shuttling SR proteins (9G8, SRp20, and SF2/ASF) have also been proposed to serve this function (10Huang Y. Steitz J.A. Mol. Cell. 2001; 7: 899-905Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 11Huang Y. Gattoni R. Stevenin J. Steitz J.A. Mol. Cell. 2003; 11: 837-843Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 12Huang Y. Yario T.A. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9666-9670Crossref PubMed Scopus (197) Google Scholar, 23Lai M.C. Tarn W.Y. J. Biol. Chem. 2004; 279: 31745-31749Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). The SR proteins are recruited to the RNA during transcription in a phosphorylated form, and dephosphorylation may trigger the recruitment of Tap (12Huang Y. Yario T.A. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9666-9670Crossref PubMed Scopus (197) Google Scholar). Although Tap has been suggested to be recruited to most cellular mRNA through protein-protein interactions, it was originally identified as an RNA-binding protein that interacted specifically with a constitutive transport element (CTE) (24Gruter P. Tabernero C. von K.C. Schmitt C. Saavedra C. Bachi A. Wilm M. Felber B.K. Izaurralde E. Mol. Cell. 1998; 1: 649-659Abstract Full Text Full Text PDF PubMed Scopus (475) Google Scholar) present in the genomic RNA of Mason-Pfizer monkey virus (MPMV) (25Bray M. Prasad S. Dubay J.W. Hunter E. Jeang K.T. Rekosh D. Hammarskjold M.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1256-1260Crossref PubMed Scopus (364) Google Scholar, 26Ernst R. Bray M. Rekosh D. Hammarskjöld M.-L. Mol. Cell Biol. 1997; 17: 135-144Crossref PubMed Scopus (136) Google Scholar, 27Hammarskjold M.L. Curr. Top. Microbiol. Immunol. 2001; 259: 77-93PubMed Google Scholar). Furthermore, we have recently demonstrated that the Tap gene, itself, also contains a functional Tap-binding CTE, which exists in a retained intron (28Li Y. Bor Y.C. Misawa Y. Xue Y. Rekosh D. Hammarskjold M.L. Nature. 2006; 443: 234-237Crossref PubMed Scopus (98) Google Scholar). An mRNA that retains this intron is exported to the cytoplasm and is translated into a small alternative protein isoform of Tap. Mutation of the Tap-binding loop of the Tap-CTE provides further evidence that Tap regulates its own expression through a mechanism that involves direct RNA binding. This clearly demonstrates that Tap is capable of binding directly to cellular mRNA. MPMV, like all other retroviruses, produces an unspliced mRNA with a retained complete intron that has to be efficiently exported and translated in the cytoplasm (25Bray M. Prasad S. Dubay J.W. Hunter E. Jeang K.T. Rekosh D. Hammarskjold M.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1256-1260Crossref PubMed Scopus (364) Google Scholar, 26Ernst R. Bray M. Rekosh D. Hammarskjöld M.-L. Mol. Cell Biol. 1997; 17: 135-144Crossref PubMed Scopus (136) Google Scholar, 27Hammarskjold M.L. Curr. Top. Microbiol. Immunol. 2001; 259: 77-93PubMed Google Scholar, 29Hammarskjold M.L. Semin. Cell Dev. Biol. 1997; 8: 83-90Crossref PubMed Scopus (42) Google Scholar). This mode of genetic organization and gene expression presents a general problem for retroviruses, because mRNAs usually remain in the nucleus until all introns are removed (30Chang D.D. Sharp P.A. Cell. 1989; 59: 789-795Abstract Full Text PDF PubMed Scopus (392) Google Scholar, 31Legrain P. Rosbash M. Cell. 1989; 57: 573-583Abstract Full Text PDF PubMed Scopus (322) Google Scholar). In complex retroviruses, such as HIV, the problem is solved by the expression of specific viral proteins (Rev in the case of HIV) that interact with cis-acting RNA elements in the viral genome (RRE in the case of HIV) (32Hadzopoulou-Cladaras M. Felber B.K. Cladaras C. Athanassopoulos A. Tse A. Pavlakis G.N. J. Virol. 1989; 63: 1265-1274Crossref PubMed Google Scholar, 33Hammarskjold M.L. Heimer J. Hammarskjold B. Sangwan I. Albert L. Rekosh D. J. Virol. 1989; 63: 1959-1966Crossref PubMed Google Scholar, 34Malim M.H. Hauber J. Le S.V. Maizel J.V. Cullen B.R. Nature. 1989; 338: 254-257Crossref PubMed Scopus (953) Google Scholar, 35Pollard V.W. Malim M.H. Annu. Rev. Microbiol. 1998; 52: 491-532Crossref PubMed Scopus (586) Google Scholar). In HIV infection, the Rev-RRE RNA complex is exported with the help of the Crm1 cellular export receptor (36Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (1744) Google Scholar, 37Neville M. Stutz F. Lee L. Davis L.I. Rosbash M. Curr. Biol. 1997; 7: 767-775Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). MPMV, however, does not encode any regulatory proteins. Instead, the CTE interacts directly with the Tap export protein and another cellular protein, NXT1/p15 (hereafter referred to as NXT1), to promote export (38Braun I.C. Herold A. Rode M. Conti E. Izaurralde E. J. Biol. Chem. 2001; 276: 20536-20543Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 39Fribourg S. Braun I.C. Izaurralde E. Conti E. Mol. Cell. 2001; 8: 645-656Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 40Guzik B.W. Levesque L. Prasad S. Bor Y.C. Black B.E. Paschal B.M. Rekosh D. Hammarskjold M.L. Mol. Cell Biol. 2001; 21: 2545-2554Crossref PubMed Scopus (87) Google Scholar, 41Katahira J. Strasser K. Podtelejnikov A. Mann M. Jung J.U. Hurt E. EMBO J. 1999; 18: 2593-2609Crossref PubMed Scopus (344) Google Scholar, 42Levesque L. Guzik B. Guan T. Coyle J. Black B.E. Rekosh D. Hammarskjold M.L. Paschal B.M. J. Biol. Chem. 2001; 276: 44953-44962Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 43Wiegand H.L. Coburn G.A. Zeng Y. Kang Y. Bogerd H.P. Cullen B.R. Mol. Cell Biol. 2002; 22: 245-256Crossref PubMed Scopus (101) Google Scholar). We have also shown that Tap/NXT1 enhances translation of CTE-containing RNA in the cytoplasm. Tap, but not NXT1, remained associated with polyribosomes (44Jin L. Guzik B.W. Bor Y.C. Rekosh D. Hammarskjold M.L. Genes Dev. 2003; 17: 3075-3086Crossref PubMed Scopus (81) Google Scholar). These results suggest that productive nuclear export of Tap-RNA complexes might be coupled to translation initiation. Here we show that moderate SRp20 or 9G8 overexpression significantly stimulates translation from unspliced RNA containing a CTE. Using 9G8, we show that RNA export is not affected, but that SR protein expression enhances polyribosome association of RNA containing the CTE. Furthermore, we show that 9G8 is present in 80 S monosomes and small polyribosomes and that hyperphosphorylated forms of this protein are significantly enriched in these fractions. These results suggest further links between RNA export and translation and highlight the complexity of post-transcriptional gene regulation. Plasmids and Cloning Procedures—To facilitate identification, all of the plasmids used in this study were indexed as numbers in the form pHRXXXX. The subgenomic HIV-1 reporter constructs, pCMVGagPol-CTE (pHR1361) (45Srinivasakumar N. Chazal N. Helga M.C. Prasad S. Hammarskjold M.L. Rekosh D. J. Virol. 1997; 71: 5841-5848Crossref PubMed Google Scholar), pCMVGagPol-TapCTE × 1 (pHR3405) (28Li Y. Bor Y.C. Misawa Y. Xue Y. Rekosh D. Hammarskjold M.L. Nature. 2006; 443: 234-237Crossref PubMed Scopus (98) Google Scholar), and pCMVGagPol-TapCTE × 2 (pHR3406) (28Li Y. Bor Y.C. Misawa Y. Xue Y. Rekosh D. Hammarskjold M.L. Nature. 2006; 443: 234-237Crossref PubMed Scopus (98) Google Scholar); a plasmid that expresses secreted alkaline phosphatase (SEAP), pCMVSEAP (pHR1831) (46Cullen B.R. Malim M.H. Methods Enzymol. 1992; 216: 362-368Crossref PubMed Scopus (164) Google Scholar); a plasmid that expresses a FLAG-tagged NXT1 protein, pcDNANXT1 (pHR2283) (47Black B.E. Levesque L. Holaska J.M. Wood T.C. Paschal B.M. Mol. Cell Biol. 1999; 19: 8616-8624Crossref PubMed Scopus (75) Google Scholar); and a plasmid that expresses a FLAG-tagged Tap protein, pcDNAFLAG-Tap (pHR2352) (44Jin L. Guzik B.W. Bor Y.C. Rekosh D. Hammarskjold M.L. Genes Dev. 2003; 17: 3075-3086Crossref PubMed Scopus (81) Google Scholar) have been described previously. Plasmids expressing T7-tagged SR proteins, pCGT79G8 (pHR2957) and pCGT7SRp20 (pHR2959) (48Caceres J.F. Misteli T. Screaton G.R. Spector D.L. Krainer A.R. J. Cell Biol. 1997; 138: 225-238Crossref PubMed Scopus (327) Google Scholar), were kind gifts from Dr. Adrian Krainer. PGEM7ZfSEAP (pHR2653), the template used to prepare in vitro transcribed seap, was constructed by inserting the BamHI/SacI fragment of pCMVSEAP (pHR1831) into pGEM7Zf (Promega, Madison, WI). This plasmid was linearized with SacI to provide T7 polymerase a template for in vitro transcription. pGEXT2ZnK (pHR3035) was constructed by PCR amplification of pCGT79G8 (pHR2957) using oligonucleotide 5′-CGCGGGATCCGAAGATGCAGTACGAGGAC-3′ as the sense primer and oligonucleotide 5′-CGCGGAATTCGTAACGATGACAATCATAAGC-3′ as the antisense primer to obtain a fragment encoding the zinc knuckle domain of 9G8. This fragment was digested with BamHI/EcoRI and ligated into pGEXT2 (AMRAD). Cell Lines and Transient Transfections—293T/17 cells (49Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2306) Google Scholar) and B2.23 cells were maintained in Iscove's modified Dulbecco's medium supplemented with 10% bovine calf serum. B2.23 cells are 293T/17 cells stably transfected with pCMVGagPol-CTE and pCMVEnv-CTE. They are similar to the previously described B2.10 cells (44Jin L. Guzik B.W. Bor Y.C. Rekosh D. Hammarskjold M.L. Genes Dev. 2003; 17: 3075-3086Crossref PubMed Scopus (81) Google Scholar, 50Coyle J.H. Guzik B.W. Bor Y.C. Jin L. Eisner-Smerage L. Taylor S.J. Rekosh D. Hammarskjold M.L. Mol. Cell Biol. 2003; 23: 92-103Crossref PubMed Scopus (92) Google Scholar). 293T/17 cells were transfected using a calcium phosphate transfection protocol (51Graham F.L. van der Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6499) Google Scholar). p24 Enzyme-linked Immunosorbent Assay and SEAP Quantitation—Supernatants from transfected cells were collected at 65-72 h post-transfection and centrifuged briefly to remove residual cells and debris. Expression levels of p24 (HIV capsid protein) were determined by an enzyme-linked immunosorbent assay protocol using a p24 monoclonal antibody (183-H12-5C) and pooled human anti-HIV immunoglobulin G (52Wehrly K. Chesebro B. Methods. 1997; 12: 288-293Crossref PubMed Scopus (122) Google Scholar). The p24 antibody was obtained from the AIDS Research and Reference Reagent Program and was contributed by Bruce Chesebro (NIAID, National Institutes of Health-Rocky Mountain Laboratories). SEAP activity in the supernatants was measured with the Phospha-Light Chemiluminescent Reporter Kit (Tropix). Western Blot Analysis and Antibodies—Proteins were separated by SDS-15% PAGE (acrylamide/bisacrylamide ratio: 30/0.14). Western blot analysis was performed essentially as previously described (33Hammarskjold M.L. Heimer J. Hammarskjold B. Sangwan I. Albert L. Rekosh D. J. Virol. 1989; 63: 1959-1966Crossref PubMed Google Scholar). Briefly, proteins were transferred to an Immobilon-P membrane (Millipore), and the membrane was blocked in 5% milk (or 5% bovine serum albumin for phospho-blots) and probed with antibody. For detection of T79G8 and T7SRp20, blots were probed with anti-T7 antibody (1:1000, Novagen). For detection of phospho-SR proteins, blots were probed with monoclonal antibody 104 (mAb104, 1:25, ATCC) (54Roth M.B. Murphy C. Gall J.G. J. Cell Biol. 1990; 111: 2217-2223Crossref PubMed Scopus (158) Google Scholar). To detect endogenous 9G8, blots were probed with a polyclonal 9G8 antibody raised in rabbits (1:250, BioSource) against a GST-9G8 zinc knuckle domain (amino acids 61-122) fusion protein produced from the pGEX2TZnK vector (pHR3035). After washing, blots were incubated with goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:5,000, Amersham Biosciences), and proteins were visualized using ECL (Amersham Biosciences). Quantitation was performed with ImageQuaNT analysis software. Polyribosome Analysis—Untransfected or transfected (48 h post-transfection) cells (4 × 107) were exposed to 50 μg/ml cycloheximide at 37 °C for 30 min (RNA analysis) or 5 min (protein analysis), washed twice with cold phosphate-buffered saline containing 50 μg/ml cycloheximide, and harvested by scraping from the plate. Pelleted cells were resuspended in 250 μl of cold RSB (10 mm Tris-HCl at pH 7.4, 10 mm NaCl, 3 mm MgCl2, protease and phosphatase inhibitor cocktails (Sigma), and 250 units of RNasin (Promega)) and lysed by the addition of an equal volume of 2× lysis buffer (RSB containing 1% Triton X-100, 1% deoxycholate, and 2% Tween 20) with incubation for 10 min on ice. Lysates were centrifuged for 10 min at 10,000 × g, and supernatants were loaded onto gradients of 10% to 50% (w/v) sucrose in 10 mm Tris-HCl (pH 7.6), 75 mm KCl, and 3 mm MgCl2. In some experiments, before loading onto the gradient, EDTA was added to the lysate at a final concentration of 15 mm to disrupt the polysomes (55Calzone F.J. Angerer R.C. Gorovsky M.A. Nucleic Acids Res. 1982; 10: 2145-2161Crossref PubMed Scopus (21) Google Scholar, 56Johannes G. Sarnow P. RNA (N. Y.). 1998; 4: 1500-1513Crossref PubMed Scopus (223) Google Scholar). After centrifugation at 36,000 rpm for 2 h at 4 °C in an SW41Ti rotor, the gradient was collected from the top using a Piston Gradient Fractionator (Biocomp) into 13 or 20 fractions, with the A254 continuously measured using a UV-M II monitor (Amersham Biosciences) during collection. Fractions were either isopropanol-precipitated or immunoprecipitated prior to SDS-PAGE, or they were purified for RNA. For the immunoprecipitations, fractions were diluted 1:2 in Tris-buffered saline (50 mm Tris-HCl, 150 mm NaCl, pH 7.4) and incubated 1 h with 20 μl of anti-T7 conjugated agarose (Bethyl Laboratories) at 4 °C with tumbling. For the isopropanol precipitations, an equal volume of ice-cold isopropanol was added to each fraction and incubated on ice 30 min. The precipitate was pelleted at 13,000 rpm for 15 min and washed in cold acetone. To purify RNA from the sucrose, 10 ng of in vitro transcribed seap RNA (produced using T7 polymerase and an SacI-digested pHR2653 template) was first added to 150-μl aliquots of each fraction and served as a control for recovery and RNA degradation. Samples were treated with 0.2 mg/ml proteinase K in 1% SDS for 30 min at 42 °C. This was followed by phenol chloroform isoamyl alcohol extraction. Next, 2.5 volumes of 100% ethanol was added, and the mixture was stored at -70 °C for 15 min. The fractions were centrifuged at 13,000 rpm for 30 min. RNA pellets were washed with 70% ethanol, resuspended in 4 μl of water with 14 μl of RS buffer (1.3× MOPS (10× MOPS, 200 mm MOPS, pH 7.0, 50 mm sodium acetate, 1 mm EDTA), 9.25% formaldehyde, 71.4% formamide), incubated at 55 °C for 10 min, cooled on ice, and loaded onto a 1% agarose gel. Northern analysis was carried out by standard procedure, as described below. RNA Fractionation and Northern Blot Analysis—The methods used for nuclear and cytoplasmic RNA extraction, poly(A)+ mRNA selection, and Northern blot analysis were previously described (33Hammarskjold M.L. Heimer J. Hammarskjold B. Sangwan I. Albert L. Rekosh D. J. Virol. 1989; 63: 1959-1966Crossref PubMed Google Scholar, 57Hammarskjöld M.L. Li H. Rekosh D. Prasad S. J. Virol. 1994; 68: 951-958Crossref PubMed Google Scholar). 293T cells were harvested at 65 h post-transfection. An SacI-BglII fragment of pHR146 containing nucleotides 682-2093 of the HIV-1 BH10 clone and a BamHI fragment of pHR1831 containing the human SEAP cDNA (nucleotides 213-1698) were labeled with [α-32P]dCTP by using the T7 Quickprime kit (Amersham Biosciences). Northern blots were quantitated with a Molecular Dynamics PhosphorImager and ImageQuaNT analysis software. Phosphatase Treatment of 9G8—Sucrose gradient fractions were either left untreated or treated with 1 μl of highly active recombinant calf intestinal alkaline phosphatase (140 units/μl, Roche Applied Science) for 30 min at 37 °C. Fractions were immunoprecipitated using anti-T7-conjugated agarose and analyzed by Western blot. Two Shuttling SR Proteins Enhance Expression from Unspliced RNA Containing the MPMV-CTE—SRp20 and 9G8, two of the shuttling SR proteins, have been reported to bind to Tap in the nucleus and contribute to Tap-mediated RNA export (10Huang Y. Steitz J.A. Mol. Cell. 2001; 7: 899-905Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 11Huang Y. Gattoni R. Stevenin J. Steitz J.A. Mol. Cell. 2003; 11: 837-843Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 12Huang Y. Yario T.A. Steitz J.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9666-9670Crossref PubMed Scopus (197) Google Scholar,23Lai M.C. Tarn W.Y. J. Biol. Chem. 2004; 279: 31745-31749Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Because Tap binds to the MPMV-CTE, and we have previously demonstrated that moderate overexpression of Tap and NXT1 increases translation from an RNA containing the CTE in 293T cells (44Jin L. Guzik B.W. Bor Y.C. Rekosh D. Hammarskjold M.L. Genes Dev. 2003; 17: 3075-3086Crossref PubMed Scopus (81) Google Scholar), we decided to test the effect of these SR proteins on CTE-mediated expression. In these studies, we utilized a CTE reporter construct (pCMVGagPol-CTE) that encodes the HIV Gag and GagPol proteins from an mRNA that remains unspliced. This mRNA is efficiently exported because of the presence of the CTE, but it is inefficiently translated in 293T cells (44Jin L. Guzik B.W. Bor Y.C. Rekosh D. Hammarskjold M.L. Genes Dev. 2003; 17: 3075-3086Crossref PubMed Scopus (81) Google Scholar, 50Coyle J.H. Guzik B.W. Bor Y.C. Jin L. Eisner-Smerage L. Taylor S.J. Rekosh D. Hammarskjold M.L. Mol. Cell Biol. 2003; 23: 92-103Crossref PubMed Scopus (92) Google Scholar). Expression of the Gag and GagPol proteins from the mRNA results in the release of virus-like particles into the medium of transfected cells. This can be quantified by an enzyme-linked immunosorbent assay measuring the HIV p24 protein. In these assays, cells are also transfected with a plasmid expressing SEAP (pCMVSEAP) from a spliced RNA. SEAP activity is measured in the supernatant and serves as a control for transfection efficiency, potential promoter competition and other "nonspecific" effects. We have utilized this assay in many previous studies of CTE function (28Li Y. Bor Y.C. Misawa Y. Xue Y. Rekosh D. Hammarskjold M.L. Nature. 2006; 443: 234-237Crossref PubMed Scopus (98) Google Scholar, 40Guzik B.W. Levesque L. Prasad S. Bor Y.C. Black B.E. Paschal B.M. Rekosh D. Hammarskjold M.L. Mol. Cell Biol. 2001; 21: 2545-2554Crossref PubMed Scopus (87) Google Scholar, 44Jin L. Guzik B.W. Bor Y.C. Rekosh D. Hammarskjold M.L. Genes Dev. 2003; 17: 3075-3086Crossref PubMed Scopus (81) Google Scholar, 50Coyle J.H. Guzik B.W. Bor Y.C. Jin L. Eisner-Smerage L. Taylor S.J. Rekosh D. Hammarskjold M.L. Mol. Cell Biol. 2003; 23: 92-103Crossref PubMed Scopus (92) Google Scholar). Fig. 1A shows that expression of 9G8 or SRp20 in conjunction with the CTE reporter in 293T cells resulted in a significant enhancement of p24 expression but had no significant effects on SEAP levels (Fig. 1B). In the experiment shown, 1 μg of the SR-protein plasmids were used together with 5 μg and 0.25 μg of the CTE and SEAP reporter plasmids, respectively, to transfect 3 × 106 cells. Quantitative Western blot analysis using 9G8-specific antibodies (see Fig. 6) indicated that this led to ∼10-fold overexpression of 9G8 (data not shown). However, a similar enhancement was seen with as little as 100 ng of the SR plasmids (data not shown). Thus, moderate overexpression of either 9G8 or SRp20 specifically increased CTE function without significant effects on SEAP expression.FIGURE 6Development of 9G8-specific antibodies. A, a schematic diagram of the 9G8 protein is shown with its RNA recognition motif (RRM), the zinc knuckle domain (ZnK) and the RS domain. The region of 9G8 cDNA encoding the unique zinc knuckle that was fused to the GST coding sequence and expressed in Escherichia coli is also shown. Purified fusion protein was used to produce polyclonal antibodies in rabbits. B and C, detection of T79G8 and T7SRp20 using an anti-T7 antibody and an anti-ZnK (9G8) antibody. 293T cells were transfected with pCGT7SRp20, pCGT79G8, or pCMV and harvested 48 h post transfection. Proteins were separated by SDS-PAGE and subjected to Western blot analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To demonstrate that the effect of the SR proteins was not limited to expression in transient transfections, we repeated the experiments using B2.23 cells, a 293T cell line that is constitutively expressing p24 from an integrated CMVGagPol-CTE plasmid. Because the effects of SRp20 and 9G8 on CTE expression were very similar, and these closely related SR proteins have been shown to have "overlapping" functions, we chose to perform the experiments with one of the SR proteins, 9G8. Transfection of B2.23 cells with the plasmid expressing 9G8 resulted in a significant (almost 10-fold) enhancement of p24 expression (Fig. 1C). As before, the cells were co-transfected with the SEAP-expressing plasmid (Fig. 1D). 9G8 had no significant effect on SEAP expression, confirming the results from the original experiments.

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