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• W2080459032 abstract "In this study, we sought to investigate the mechanism by which heterogeneous nuclear ribonucleoprotein (hnRNP) H and F regulate proteolipid protein (PLP)/DM20 alternative splicing. G-rich sequences in exon 3B, G1 and M2, are required for hnRNPH- and F-mediated regulation of the PLP/DM20 ratio and, when placed between competing 5′ splice sites in an α-globin minigene, direct hnRNPH/F-regulated alternative splicing. In contrast, the activity of the intronic splicing enhancer, which is necessary for PLP splicing, is only modestly reduced by removal of hnRNPH/F both in PLP and α-globin gene context. In vivo, hnRNPH reversed reduction of DM20 splicing induced by hnRNPH/F removal, whereas hnRNPF had little effect. Tethering of the MS2-hnRNPH fusion protein downstream of the DM20 5′ splice site increased DM20 splicing, whereas MS2-hnRNPF did not. Binding of U1 small nuclear ribonucleoprotein (U1snRNP) to DM20 is greatly impaired by mutation of G1 and M2 and depletion of hnRNPH and F. Reconstitution of hnRNPH/F-depleted extracts with either hnRNPH or F restored U1snRNP binding. We conclude that hnRNPH and F regulate DM20 splicing by recruiting U1snRNP and that hnRNPH plays a primary role in DM20 splice site selection in vivo. Decreased expression of hnRNPH/F in differentiated oligodendrocytes may regulate the PLP/DM20 ratio by reducing DM20 5′ splice site recognition by U1snRNP. In this study, we sought to investigate the mechanism by which heterogeneous nuclear ribonucleoprotein (hnRNP) H and F regulate proteolipid protein (PLP)/DM20 alternative splicing. G-rich sequences in exon 3B, G1 and M2, are required for hnRNPH- and F-mediated regulation of the PLP/DM20 ratio and, when placed between competing 5′ splice sites in an α-globin minigene, direct hnRNPH/F-regulated alternative splicing. In contrast, the activity of the intronic splicing enhancer, which is necessary for PLP splicing, is only modestly reduced by removal of hnRNPH/F both in PLP and α-globin gene context. In vivo, hnRNPH reversed reduction of DM20 splicing induced by hnRNPH/F removal, whereas hnRNPF had little effect. Tethering of the MS2-hnRNPH fusion protein downstream of the DM20 5′ splice site increased DM20 splicing, whereas MS2-hnRNPF did not. Binding of U1 small nuclear ribonucleoprotein (U1snRNP) to DM20 is greatly impaired by mutation of G1 and M2 and depletion of hnRNPH and F. Reconstitution of hnRNPH/F-depleted extracts with either hnRNPH or F restored U1snRNP binding. We conclude that hnRNPH and F regulate DM20 splicing by recruiting U1snRNP and that hnRNPH plays a primary role in DM20 splice site selection in vivo. Decreased expression of hnRNPH/F in differentiated oligodendrocytes may regulate the PLP/DM20 ratio by reducing DM20 5′ splice site recognition by U1snRNP. Alternative splicing of a single transcript is widely utilized to generate proteomic diversity in response to developmental, cell specific, and external signals (1.Modrek B. Lee C.J. Nat. Genet. 2003; 34: 177-180Crossref PubMed Scopus (425) Google Scholar). Typically, alternatively spliced sites are weak and the final splicing selection depends on the interplay of enhancers and silencers and the relative abundance and/or affinity of the RNA binding factors (2.Black D.L. Annu. Rev. Biochem. 2003; 72: 291-336Crossref PubMed Scopus (1947) Google Scholar, 3.Hertel K.J. J. Biol. Chem. 2008; 283: 1211-1215Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Early recognition of the 5′ splice sites is mediated by the U1snRNP 2The abbreviations used are: U1snRNP, U1 small nuclear ribonucleoprotein; U1snRNA, U1 small nuclear RNA; hnRNP, heterogeneous nuclear ribonucleoprotein; PLP, proteolipid protein; siRNA, small interference RNA; RT, reverse transcription; MS, MS2 coat protein binding motif; NR, non-related sequence; WT, wild type; MT, mutated; Bt2cAMP, dibutyryl cyclic AMP; ISE, intronic splicing enhancer. 2The abbreviations used are: U1snRNP, U1 small nuclear ribonucleoprotein; U1snRNA, U1 small nuclear RNA; hnRNP, heterogeneous nuclear ribonucleoprotein; PLP, proteolipid protein; siRNA, small interference RNA; RT, reverse transcription; MS, MS2 coat protein binding motif; NR, non-related sequence; WT, wild type; MT, mutated; Bt2cAMP, dibutyryl cyclic AMP; ISE, intronic splicing enhancer. through direct base pairing of the single-stranded 5′-end of U1snRNA with six conserved nucleotides at the 5′ splice site. Binding of U1snRNP and base pairing of the U1snRNA to the template is required for spliceosome assembly (4.Green M.R. Annu. Rev. Cell Biol. 1991; 7: 559-599Crossref PubMed Scopus (553) Google Scholar, 5.Seraphin B. Rosbash M. Cell. 1989; 59: 349-358Abstract Full Text PDF PubMed Scopus (269) Google Scholar).A number of splicing factors that bind to either enhancers or silencers have been identified and shown to influence the efficiency of splice site recognition and spliceosome assembly (6.Matlin A.J. Clark F. Smith C.W. Nat. Rev. Mol. Cell Biol. 2005; 6: 386-398Crossref PubMed Scopus (959) Google Scholar). The hnRNPs are a large family of ubiquitously expressed RNA binding factors, which, in addition to regulating constitutive splicing, play an important role in alternative splicing (7.Han K. Yeo G. An P. Burge C.B. Grabowski P.J. PLoS Biol. 2005; 3: e158Crossref PubMed Scopus (128) Google Scholar, 8.Krecic A.M. Swanson M.S. Curr. Opin. Cell Biol. 1999; 11: 363-371Crossref PubMed Scopus (708) Google Scholar). hnRNPH and F are highly homologous proteins that bind to G-rich sequences present in exons, in introns, and in close proximity to the polyadenylation site (8.Krecic A.M. Swanson M.S. Curr. Opin. Cell Biol. 1999; 11: 363-371Crossref PubMed Scopus (708) Google Scholar, 9.Caputi M. Zahler A.M. J. Biol. Chem. 2001; 276: 43850-43859Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 10.Marcucci R. Baralle F.E. Romano M. Nucleic Acids Res. 2007; 35: 132-142Crossref PubMed Scopus (26) Google Scholar, 11.Matunis M.J. Xing J. Dreyfuss G. Nucleic Acids Res. 1994; 22: 1059-1067Crossref PubMed Scopus (128) Google Scholar, 12.Veraldi K.L. Arhin G.K. Martincic K. Chung-Ganster L.H. Wilusz J. Milcarek C. Mol. Cell. Biol. 2001; 21: 1228-1238Crossref PubMed Scopus (109) Google Scholar). Depending on the gene context, these splicing factors have different affinity for their cognate sequences and appear to act in concert to regulate splicing of a number of genes (13.Alkan S.A. Martincic K. Milcarek C. Biochem. J. 2006; 393: 361-371Crossref PubMed Scopus (48) Google Scholar, 14.Caputi M. Zahler A.M. EMBO J. 2002; 21: 845-855Crossref PubMed Scopus (103) Google Scholar). Although they are most often inhibitors of alternatively spliced exons, they can also function as enhancers (14.Caputi M. Zahler A.M. EMBO J. 2002; 21: 845-855Crossref PubMed Scopus (103) Google Scholar, 15.Chen C.D. Kobayashi R. Helfman D.M. Genes Dev. 1999; 13: 593-606Crossref PubMed Scopus (167) Google Scholar, 16.Chou M.Y. Rooke N. Turck C.W. Black D.L. Mol. Cell. Biol. 1999; 19: 69-77Crossref PubMed Scopus (213) Google Scholar, 17.Garneau D. Revil T. Fisette J.F. Chabot B. J. Biol. Chem. 2005; 280: 22641-22650Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 18.Min H. Chan R.C. Black D.L. Genes Dev. 1995; 9: 2659-2671Crossref PubMed Scopus (169) Google Scholar). G-rich sequences are highly conserved within introns in close proximity to splice sites (7.Han K. Yeo G. An P. Burge C.B. Grabowski P.J. PLoS Biol. 2005; 3: e158Crossref PubMed Scopus (128) Google Scholar, 10.Marcucci R. Baralle F.E. Romano M. Nucleic Acids Res. 2007; 35: 132-142Crossref PubMed Scopus (26) Google Scholar, 19.Nussinov R. J. Theor. Biol. 1988; 133: 73-84Crossref PubMed Scopus (25) Google Scholar, 20.Nussinov R. J. Biomol. Struct. Dyn. 1989; 6: 985-1000Crossref PubMed Scopus (19) Google Scholar, 21.Sirand-Pugnet P. Durosay P. Brody E. Marie J. Nucleic Acids Res. 1995; 23: 3501-3507Crossref PubMed Scopus (106) Google Scholar). In genes containing short introns, these G-rich sequences allow splice site recognition through an intron-definition mechanism (22.Carlo T. Sterner D.A. Berget S.M. RNA. 1996; 2: 342-353PubMed Google Scholar, 23.McCullough A.J. Berget S.M. Mol. Cell. Biol. 1997; 17: 4562-4571Crossref PubMed Scopus (183) Google Scholar). In an artificial α-globin gene construct containing duplicated 5′ splice sites flanked by identical G triplets, the G-rich sequences favor selection of the distal 5′ splice site through binding of U1snRNA to the G-rich sequence by direct base pairing (24.McCullough A.J. Berget S.M. Mol. Cell. Biol. 2000; 20: 9225-9235Crossref PubMed Scopus (72) Google Scholar).The proteolipid protein (PLP) gene gives rise to two isoforms, PLP and DM20, through alternative splicing of competing 5′ splice sites resulting in either inclusion or exclusion of exon 3B. G-rich enhancers, named M2 and ISE, flank the DM20 and PLP 5′ splice sites, respectively (25.Hobson G.M. Huang Z. Sperle K. Stabley D.L. Marks H.G. Cambi F. Ann. Neurol. 2002; 52: 477-488Crossref PubMed Scopus (43) Google Scholar, 26.Nave K.A. Lai C. Bloom F.E. Milner R.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5665-5669Crossref PubMed Scopus (267) Google Scholar, 27.Wang E. Dimova N. Cambi F. Nucleic Acids Res. 2007; 35: 4164-4178Crossref PubMed Scopus (56) Google Scholar). In addition, there are other G-rich sequences in exon 3B, and all are potential hnRNPH/F binding motifs (27.Wang E. Dimova N. Cambi F. Nucleic Acids Res. 2007; 35: 4164-4178Crossref PubMed Scopus (56) Google Scholar, 28.Schaub M.C. Lopez S.R. Caputi M. J. Biol. Chem. 2007; 282: 13617-13626Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The PLP/DM20 ratio increases in postnatal brain development and in differentiated oligodendrocytes, and this is temporally related to reduced expression of hnRNPH and F (27.Wang E. Dimova N. Cambi F. Nucleic Acids Res. 2007; 35: 4164-4178Crossref PubMed Scopus (56) Google Scholar, 29.Wang E. Dimova N. Sperle K. Huang Z. Lock L. McCulloch M.C. Edgar J.M. Hobson G.M. Cambi F. Exp. Neurol. 2008; 214: 322-330Crossref PubMed Scopus (21) Google Scholar). The DM20 isoform is preferentially expressed in embryonic brain and non-glial cells (30.LeVine S.M. Wong D. Macklin W.B. Dev. Neurosci. 1990; 12: 235-250Crossref PubMed Scopus (78) Google Scholar). Knockdown of hnRNPH and F synergistically increased the PLP/DM20 ratio, and this effect was mediated by M2 in concert with some or all of the G runs present in PLP exon 3B, whereas the ISE had little contribution to this regulation (27.Wang E. Dimova N. Cambi F. Nucleic Acids Res. 2007; 35: 4164-4178Crossref PubMed Scopus (56) Google Scholar). hnRNPH and F have been shown to serve redundant functions in regulating alternative splicing and to cooperatively regulate other genes (17.Garneau D. Revil T. Fisette J.F. Chabot B. J. Biol. Chem. 2005; 280: 22641-22650Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 31.Crawford J.B. Patton J.G. Mol. Cell. Biol. 2006; 26: 8791-8802Crossref PubMed Scopus (39) Google Scholar, 32.Mauger D.M. Lin C. Garcia-Blanco M.A. Mol. Cell. Biol. 2008; 28: 5403-5419Crossref PubMed Scopus (89) Google Scholar); however, to the best of our knowledge, a synergistic effect such as that observed with PLP alternative splicing was not previously reported.In this study, we sought to investigate further the mechanism by which hnRNPH and F regulate the PLP/DM20 ratio. A proximal G-rich sequence (G1) and M2 are required for hnRNPH- and F-mediated regulation of the PLP/DM20 ratio. In contrast, the ISE enhancer's activity is only modestly reduced by removal of hnRNPH/F. In vivo, hnRNPH and F are not functionally redundant, and hnRNPH plays a primary role in regulating the PLP/DM20 ratio. In vitro, hnRNPH and hnRNPF were able to restore binding of U1snRNP, which was greatly reduced by removal of hnRNPH/F. The data show that both hnRNPH and F regulate DM20 splicing by recruiting U1snRNP and suggest that hnRNPH may have additional functions in regulating the splicing machinery.EXPERIMENTAL PROCEDURESPlasmids—The pcDNA-MS2-H and pcDNA-MS2-F plasmids expressing MS2 coat protein fused in-frame to Myc-hisA-tagged hnRNPH and F, respectively, were a generous gift of Dr. Mark McNally (33.McNally L.M. Yee L. McNally M.T. J. Biol. Chem. 2006; 281: 2478-2488Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The pFLAG-hnRNPH and pFLAG-hnRNPF plasmids were a generous gift of Dr. Mariano Garcia-Blanco (32.Mauger D.M. Lin C. Garcia-Blanco M.A. Mol. Cell. Biol. 2008; 28: 5403-5419Crossref PubMed Scopus (89) Google Scholar). These plasmids were used in transfections into Oli-neu cells treated with siF/H, which targets a sequence common to mouse hnRNPH and F. The human hnRNPF has a two-nucleotide mismatch with the mouse sequence targeted by the siF/H, thus it is resistant to siRNAs-mediated degradation. The siF/H-targeted sequence is identical between the human and mouse hnRNPH transcripts, and the FLAG-hnRNPH transcript is targeted by siF/H and degraded (data not shown). To make FLAG-hnRNPH resistant to siF/H degradation, we changed two nucleotides (supplemental Table S1). The SRp40 was a kind gift of Dr. Stefan Stamm. The wild-type α-globin splicing construct was a kind gift of Dr. Andrew McCullough (23.McCullough A.J. Berget S.M. Mol. Cell. Biol. 1997; 17: 4562-4571Crossref PubMed Scopus (183) Google Scholar). Mutant PLP-neo (Figs. 1, 2A, and 5) and α-globin constructs (Figs. 2B and 3A) were generated by site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). In the PLP-neo, the G runs were replaced by polyTs, as previously described (27.Wang E. Dimova N. Cambi F. Nucleic Acids Res. 2007; 35: 4164-4178Crossref PubMed Scopus (56) Google Scholar). In the α-globin construct, the G runs were mutated as previously described (23.McCullough A.J. Berget S.M. Mol. Cell. Biol. 1997; 17: 4562-4571Crossref PubMed Scopus (183) Google Scholar) or replaced by G1M2 or ISE sequences from the PLP exon 3B as indicated in Fig. 3. The distance of the ISE and G1M2 sequences relative to the α-globin 5′ splice sites was the same as that in their endogenous position in the PLP gene. The SαGLO-ISE was generated by deleting the duplicated sequence containing the G1M2 in the α-glo-G1M2-ISE, thus leaving a single α-globin 5′ splice site and the downstream intron containing ISE (Fig. 2B). The SαGLO-ISEMT1, -MT2, and -MT3 were generated by site-directed mutagenesis of the SαGLO-ISE (Fig. 2B). The PLPneo-MS1 and PLPneo-NR were made by replacing twenty-two nucleotides spanning G1, M2, and the intervening five nucleotides with twenty-two nucleotides containing the MS2 protein binding motif or an unrelated sequence (Fig. 5). The PLP-neo-MS2 (data not shown) and -MS3 were made by replacing twenty-two nucleotides spanning M4–M6 and M3–M5 with the MS2 protein binding motif, respectively, in the PLP-neo-MS1 (Fig. 5). The Bcl-x minigene construct was a kind gift of Dr. Charles Chalfant (34.Massiello A. Salas A. Pinkerman R.L. Roddy P. Roesser J.R. Chalfant C.E. J. Biol. Chem. 2004; 279: 15799-15804Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar).FIGURE 2Analysis of the ISE function in PLP and α-globin gene. A, the PLP-neo splicing construct is shown. The arrows indicate the position of the PCR primers. Partial sequences of PLP exon 3B (uppercase) and intron 3 (lowercase) in the PLP-neo are shown, DM20 and PLP 5′ splice sites are in bold, M2MT and ISE are underlined, and the putative SRp40 motif in the ISE is italicized and in bold. The mutations are shown below the ISE sequences. Representative RT-PCR analysis of M2MT-, M2MT-ISEMT1-, M2MT-ISEMT2-, and M2MT-ISEMT3-derived PLP and DM20 products amplified from RNA isolated from Oli-neu cells (n = 3) (30 PCR cycles). B, schematic of SαGLO-ISE construct is shown. The arrowheads indicate the position of primers used for PCR amplification. Partial sequences of the α-globin construct spanning the 5′ splice site, the intron, and the 3′ splice site. The ISE is underlined, and the putative SRp40 motif in the ISE is italicized and in bold. The nucleotides that replace the endogenous α-globin G4 sequence are shown in bold. The mutations are shown below the ISE. Representative RT-PCR analysis of SαGLO-ISE derived spliced and unspliced products amplified from RNA isolated from Oli-neu cells either treated with control siRNA (mock) or with siF/H (n = 2) (30 PCR cycles). Representative RT-PCR analysis of SαGLO-ISE-, SαGLO-ISEMT1-, SαGLO-ISEMT2-, and SαGLO-ISEMT3-derived spliced and unspliced products (n = 2). The ratio of unspliced/spliced products is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5hnRNPH directly promotes DM20 splicing in vivo. A, schematic of PLP-neo-WT and constructs in which sequences spanning G1 and M2 have been replaced with MS2 coat protein binding motif (PLP-neo-MS1) or an unrelated sequence (PLP-neo-NR). PLP-neo-MS3 was derived from PLP-neo-MS1 by replacing sequences spanning M3-M5 with the MS2 motif (see “Experimental Procedures”). The sequences are shown. The stem-loop represents the binding site for the MS2 coat protein. B, RT-PCR analysis of PLP and DM20 products derived from the minigenes. Oli-neu cells were transfected with 0.5 μg(+) and 1.0 μg(++) of the indicated plasmids and RNA was subjected to RT-PCR (30 PCR cycles). A representative experiment is shown (n = 2). The data are expressed as percent of the DM20 product versus the total PLP+DM20 product. C, RT-PCR analysis of PLP and DM20 products derived from PLP-neo-MS3. Oli-neu cells were transfected with 0.5 μg(+) of the indicated plasmids, and RNA was subjected to RT-PCR (30 PCR cycles). A representative experiment is shown (n = 2). The data are expressed as percent of the DM20 product versus the total PLP+DM20 product.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3hnRNPH and F regulate the proximal/distal 5′ splice site ratio of α-globin. A, partial sequences of the α-globin construct spanning the duplicated 5′ splice sites, the intron, and the 3′ acceptor splice site. The sequences introduced to generate the mutated constructs are shown below the wild-type sequences. B, schematic of all α-globin constructs is shown. The filled circles represent wild-type α-globin sequences and the open circles represent the mutated sequences. The arrowheads indicate the position of primers used for PCR amplification. C, representative RT-PCR analysis of proximal (P) and distal (D) α-globin products amplified from RNA isolated from Oli-neu cells treated with siH3, siF3, siH3+siF3, and siF/H (30 PCR cycles). Mock are cells treated with scrambled siRNA. The bar graph shows the P/D ratios ± S.E. (n = 4). *, p < 0.05 and **, p < 0.01. D, representative RT-PCR analysis of proximal (P) and distal (D) products derived from all G run mutated α-globin construct amplified from RNA isolated from Oli-neu cells treated with siF/H (30 PCR cycles). Mock are cells treated with control siRNA. The bar graph shows the P/D ratios ± S.E. (n = 4). **, p < 0.01. E, representative RT-PCR analysis of proximal (P) and distal (D) α-globin products derived from G1M2-ISE and G1M2-G1M2 α-globin constructs amplified from RNA isolated from Oli-neu cells treated with siF/H (30 PCR cycles). Mock are cells treated with scrambled siRNA. The wild-type α-globin is used as control. The bar graph shows the P/D ratios ± S.E. (n = 4). *, p < 0.05; **, p < 0.01; ns = non significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)siRNAs, Cell Cultures, and Transfections—Pre-designed double-stranded siRNAs targeting hnRNPF (ID# 175722 (siF3)), hnRNPH (ID# 75775 (siH3)), the custom-made double-stranded siRNA, siF/H (27.Wang E. Dimova N. Cambi F. Nucleic Acids Res. 2007; 35: 4164-4178Crossref PubMed Scopus (56) Google Scholar), and Silencer® Negative Control #1 siRNA were purchased from Ambion (Austin, TX). Oli-neu cells were cultured in SATO medium (Dulbecco's modified Eagle's medium containing 10 ng/ml biotin, 10 ng/ml apotransferrin, 100 μm putrescine, 2 μm progesterone, 2 μm sodium selenite, 2.5 μg/ml insulin) with 1% horse serum (27.Wang E. Dimova N. Cambi F. Nucleic Acids Res. 2007; 35: 4164-4178Crossref PubMed Scopus (56) Google Scholar). Fibroblast L cells and the neuronal Neuro2A cells (ATCC) were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Cells were co-transfected with plasmid DNAs (0.5 μg, unless otherwise indicated) and siRNAs (80 nm) using the siPORT amine transfection reagent according to the Neofection protocol (Ambion) (27.Wang E. Dimova N. Cambi F. Nucleic Acids Res. 2007; 35: 4164-4178Crossref PubMed Scopus (56) Google Scholar). Western blot analysis showed that expression of hnRNPF was not affected by treatment with siH3, and expression of hnRNPH was not affected by treatment with siF3, thus excluding compensation effects (data not shown). In overexpression studies, 0.5 μg of DNA of the splicing construct was co-transfected with 0.5, 1, and 1.5 μg of the expression plasmid DNA or 0.5 μg of the empty vector pcDNA3 using the siPORT amine transfection reagent.RNA Extraction and RT-PCR—Total RNA was extracted from cultured cells using the RNeasy Mini Kit (Qiagen, Valencia, CA) and was treated with the DNA-free Kit (Ambion) according to the manufacturer's instructions. Reverse transcription was performed with 1 μg of total RNA using random hexamer primer mixture according to the manufacturer's instructions (BD Biosciences). The PCR products derived from the wild-type and mutated PLP-neo constructs were amplified by RT-PCR using a primer set previously described (Fig. 1) (27.Wang E. Dimova N. Cambi F. Nucleic Acids Res. 2007; 35: 4164-4178Crossref PubMed Scopus (56) Google Scholar, 35.Wang E. Huang Z. Hobson G.M. Dimova N. Sperle K. McCullough A. Cambi F. J. Cell. Biochem. 2006; 97: 999-1016Crossref PubMed Scopus (17) Google Scholar). The proximal and distal splice products derived from the wild-type and mutated α-globin constructs were amplified by RT-PCR with a forward primer (5′-TGGTACCGAGCTCGGATCCGATGT-3′) and a reverse primer (5′-GATGGATATCTGCAGAATTCGGGA-3′) complementary to sequences in pcDNA3 vector (Figs. 2B and 3). The XL and XS products derived from the Bcl-x minigene construct were amplified by RT-PCR with primers described previously (36.Paronetto M.P. Achsel T. Massiello A. Chalfant C.E. Sette C. J. Cell Biol. 2007; 176: 929-939Crossref PubMed Scopus (235) Google Scholar).Nuclear Extracts, Recombinant Proteins, and RNA Affinity Precipitations—Nuclear extracts were prepared from Oli-neu differentiated in SATO medium containing 1 mm Bt2cAMP for 7 days or from Oli-neu treated with siF/H for 72 h in SATO medium without Bt2cAMP using the NEP kit (Pierce) according to the manufacturer's instructions (27.Wang E. Dimova N. Cambi F. Nucleic Acids Res. 2007; 35: 4164-4178Crossref PubMed Scopus (56) Google Scholar). Poly(G) affinity column (Sigma)-mediated removal of hnRNPH and F from nuclear extracts was performed as described (37.Carlo T. Sierra R. Berget S.M. Mol. Cell. Biol. 2000; 20: 3988-3995Crossref PubMed Scopus (41) Google Scholar) Recombinant His-tagged hnRNPF and H proteins were generated in Escherichia coli and purified as described before (16.Chou M.Y. Rooke N. Turck C.W. Black D.L. Mol. Cell. Biol. 1999; 19: 69-77Crossref PubMed Scopus (213) Google Scholar). The wild-type RNA template spans 10 nucleotides upstream and 35 nucleotides downstream of the DM20 5′ splice site and contains the DM20 5′ splice site and the G-rich enhancers, G1 and M2 (DM20-G1M2-WT, Fig. 6). The mutated RNA template is identical to the wild-type except that G1 and M2 sequences were replaced by polyUs (DM20-G1M2-MT, Fig. 6). RNA templates were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). RNA affinity precipitations were performed with biotinylated RNA templates, as previously described (27.Wang E. Dimova N. Cambi F. Nucleic Acids Res. 2007; 35: 4164-4178Crossref PubMed Scopus (56) Google Scholar) except that the complexes were not UV-cross-linked.FIGURE 6Mutations of G1 and M2 interfere with binding of U1 70K and U1A in RNA affinity precipitates. A, RNA templates used in RNA affinity precipitations. The DM20 5′ splice site is in bold italics, and the natural G1 and M2 and the mutated poly-U sequences are underlined. B, representative sypro-ruby stained gel of RNA affinity precipitates with biotinylated RNA templates containing wild-type (WT) or poly-U mutated G1M2 (MT) and Oli-neu extracts (n = 3) (“Experimental Procedures”). M = protein markers. We have analyzed by liquid chromatography-MS/MS bands that are absent and one band that is more prominent in precipitates with the G1M2 mutated template. The protein's identity is shown. C, Western blot analysis of U1 70K, U1A, hnRNPA1, F, H, and L in the RNA affinity precipitates with Oli-neu extracts and either WT or MT template. The data were repeated in three separate experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Protein Isolation and Identification by Liquid Chromatography-MS/MS and Western Blot Analysis—Proteins were eluted from the streptavidin beads in 100 μl of SDS-PAGE loading buffer at 95 °C for 5 min, and one-third of the mixtures were separated by 10% SDS-PAGE and either visualized by syproruby staining (Invitrogen) or blotted to nitrocellulose membrane. Selected bands were excised from the gel and analyzed by matrix-assisted laser desorption ionization-MS/MS and liquid chromatography-MS/MS (27.Wang E. Dimova N. Cambi F. Nucleic Acids Res. 2007; 35: 4164-4178Crossref PubMed Scopus (56) Google Scholar) (Proteomics and Mass Spectrometry Core Facility, University of Kentucky). Nitrocellulose membranes were reacted with antibodies to: U1 70K (Aviva Systems Biology, San Diego, CA), U1A (Aviva Systems Biology), hnRNPH (Bethyl Laboratories) and hnRNPHF (rabbit polyclonal, generous gift of Dr. Douglas Black), A1 and L (Abcam), SRp40 (Santa Cruz Biotechnology), Myc tag (Invitrogen), and FLAG tag (Sigma) diluted 1:2000, horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) diluted 1:2000, and developed with enhanced chemiluminescence (ECL, Amersham Biosciences). Blots were visualized with a Kodak 440CF Digital Image Station using one-dimensional analysis software.RESULTSThe Synergistic Effect of hnRNPH/F Is Mediated by G1 and M2—We have shown previously that siRNA-mediated knock-down of hnRNPH and F synergistically increased the PLP/DM20 ratio in oligodendrocytes, and this effect was in large part dependent on M2 and G runs in exon 3B, named G1, G4, and G5.To determine the G runs that are necessary for the hnRNPH/F synergism in addition to M2, we tested the impact of individual mutations of G1, G4, and G5 on the hnRNPH/F-mediated increase in PLP/DM20 ratio (Fig. 1, A and B). Oli-neu cells were transfected with the G1-MT, G4-MT, and G5-MT constructs and the plasmid-derived PLP and DM20 products were amplified by RT-PCR in RNA isolated from cells cultured for 72 h in growth medium. Although the PLP/DM20 ratio derived from G4-MT and G5-MT was 0.15 ± 0.02 and 0.1 ± 0.03, respectively, compared with 0.38 ± 0.02 with the WT construct, the PLP/DM20 ratio derived from G1-MT was 3.06 ± 0.4 (Fig. 1, C (upper panel) and D). The data suggest that G1 is an enhancer of the DM20 5′ splice site, whereas G4 and G5 appear to enhance PLP 5′ splice site selection, in keeping with the results obtained previously with M6-MT and M8-MT, mutations that encompass G4 and G5, respectively (27.Wang E. Dimova N. Cambi F. Nucleic Acids Res. 2007; 35: 4164-4178Crossref PubMed Scopus (56) Google Scholar). G1-MT increased the PLP/DM20 ratio by 8-fold compared with ∼5-fold induced by M2-MT (3.06 ± 0.40 versus 2.05 ± 0.20) (Fig. 1, C (upper panel) and D (27.Wang E. Dimova N. Cambi F. Nucleic Acids Res. 2007; 35: 4164-4178Crossref PubMed Scopus (56) Google Scholar)), suggesting that G1 is a stronger enhancer of DM20 5′ splice site than M2. For mutations of G1 and M2, G1M2-MT increased the PLP/DM20 ratio to 3.61 ± 0.8 (Fig. 1, C (upper panel) and D). Mutations of G1 and M2 combined with the ISE deletion, G1M2-MT/ISEdel, resulted in a PLP/DM20 ratio of 0.44 ± 0.06 (Fig. 1, C (upper panel) and D). This is higher than the wild-type ratio, but lower than the G1M2 mutation alone and reflects the loss of both the PLP 5′ splice site-enhancing ISE and the DM20 5′ splice site-enhancing G1 and M2 sequences. Likewise, the PLP/DM20 ratio derived from constructs in which G1M2, G4, and G5 are all mutated and the ISE is present, is 0.7 ± 0.1 and is higher than that derived from M2G1G4G5-MT/ISEdel (0.21 ± 0.01 (27.Wang E. Dimova N. Cambi F. Nucleic Acids Res. 2007; 35: 4164-4178Crossref PubMed Scopus (56) Google Scholar)), reflecting the loss of the PLP-enhancing function of G4, G5, and the ISE (see next section) (Fig. 1, C (upper panel) and D). The data indicate that, in addition to M2, G1 is an enhancer of DM20 5′ splice site selection.We next assessed whether siRNA-mediated knock-down of hnRNPH and F (siH/F) affected the PLP/DM20 ratio derived from G1-MT, G4-MT, G5-MT, and G1M2-MT in Oli-n" @default.
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• W2080459032 title "Heterogeneous Nuclear Ribonucleoproteins H and F Regulate the Proteolipid Protein/DM20 Ratio by Recruiting U1 Small Nuclear Ribonucleoprotein through a Complex Array of G Runs" @default.
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