Newly Initiated RNA Encounters a Factor Involved in Splicing Immediately upon Emerging from within RNA Polymerase II
2004; Elsevier BV; Volume: 279; Issue: 48 Linguagem: Inglês
10.1074/jbc.m409087200
ISSN1083-351X
Autores Tópico(s)RNA modifications and cancer
ResumoWe employed RNA-protein cross-linking to map the path of the nascent RNA as it emerges from within RNA polymerase II. A UV-cross-linkable uridine analog was incorporated at two positions within the first five nucleotides of the transcript. Only the two largest subunits of RNA polymerase II cross-linked to the transcript in complexes containing 17–24-nucleotide (nt) RNAs. Extension of the RNA to 26 or 28 nt revealed an additional strong cross-link to the splicing factor U2AF65. In U17 complexes, in which the RNA is still contained within the polymerase, U2AF65 is tightly bound. In contrast, U2AF65 is more loosely bound in C28 transcription complexes, in which about 10 nt of transcript have emerged from the RNA polymerase. Cross-linking of U2AF65 to RNA in a C28 complex was eliminated by the addition of an excess of an RNA oligonucleotide containing the consensus U2AF65 binding site, but U2AF65 was not displaced by a nonconsensus RNA. These findings indicate that U2AF65 shifts from protein-protein to protein-RNA interactions as the RNA emerges from the polymerase. During transcription of one particular template at low UTP concentration, RNA polymerase II pauses just after synthesizing a transcript segment that is a U2AF65 binding site. Dwell time of the polymerase at this pause site was significantly and specifically reduced by the addition of recombinant U2AF65 to the transcription reaction. Therefore, the association of U2AF65 with RNA polymerase II may function not only to deliver U2AF65 to the nascent transcript but also to modulate efficient transcript elongation. We employed RNA-protein cross-linking to map the path of the nascent RNA as it emerges from within RNA polymerase II. A UV-cross-linkable uridine analog was incorporated at two positions within the first five nucleotides of the transcript. Only the two largest subunits of RNA polymerase II cross-linked to the transcript in complexes containing 17–24-nucleotide (nt) RNAs. Extension of the RNA to 26 or 28 nt revealed an additional strong cross-link to the splicing factor U2AF65. In U17 complexes, in which the RNA is still contained within the polymerase, U2AF65 is tightly bound. In contrast, U2AF65 is more loosely bound in C28 transcription complexes, in which about 10 nt of transcript have emerged from the RNA polymerase. Cross-linking of U2AF65 to RNA in a C28 complex was eliminated by the addition of an excess of an RNA oligonucleotide containing the consensus U2AF65 binding site, but U2AF65 was not displaced by a nonconsensus RNA. These findings indicate that U2AF65 shifts from protein-protein to protein-RNA interactions as the RNA emerges from the polymerase. During transcription of one particular template at low UTP concentration, RNA polymerase II pauses just after synthesizing a transcript segment that is a U2AF65 binding site. Dwell time of the polymerase at this pause site was significantly and specifically reduced by the addition of recombinant U2AF65 to the transcription reaction. Therefore, the association of U2AF65 with RNA polymerase II may function not only to deliver U2AF65 to the nascent transcript but also to modulate efficient transcript elongation. It is now appreciated that transcription is regulated during promoter clearance and transcript elongation as well as at transcription complex assembly (recently reviewed in Refs. 1Dvir A. Bba Gene Struct. Express. 2002; 1577: 208-223Crossref PubMed Scopus (50) Google Scholar, 2Shilatifard A. Conaway R.C. Conaway J.W. Annu. Rev. Biochem. 2003; 72: 693-715Crossref PubMed Scopus (199) Google Scholar, 3Hartzog G.A. Curr. Opin. Genet. Dev. 2003; 13: 119-126Crossref PubMed Scopus (40) Google Scholar, 4Arndt K.M. Kane C.M. Trends Genet. 2003; 19: 543-550Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 5Shilatifard A. Bba Gene Struct. Express. 2004; 1677: 79-86Crossref PubMed Scopus (50) Google Scholar, 6Belotserkovskaya R. Reinberg D. Curr. Opin. Genet. Dev. 2004; 14: 139-146Crossref PubMed Scopus (105) Google Scholar). Also, it is becoming increasingly apparent that transcription and RNA processing are both interconnected and interdependent (recent reviews include Refs. 7Proudfoot N.J. Furger A. Dye M.J. Cell. 2002; 108: 501-512Abstract Full Text Full Text PDF PubMed Scopus (841) Google Scholar, 8Maniatis T. Reed R. Nature. 2002; 416: 499-506Crossref PubMed Scopus (925) Google Scholar, 9Zorio D.A.R. Bentley D.L. Exp. Cell Res. 2004; 296: 91-97Crossref PubMed Scopus (115) Google Scholar). It is thus of considerable importance to carefully characterize the interaction of the transcription and processing machineries. We now have a detailed picture of RNA polymerase II structure (10Armache K.-J. Kettenberger H. Cramer P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6964-6968Crossref PubMed Scopus (195) Google Scholar, 11Bushnell D.A. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6969-6973Crossref PubMed Scopus (222) Google Scholar), but how factors interact with this structure to modulate elongation and RNA processing is still very poorly understood. In particular, it is not certain how the transcript itself interacts with RNA polymerase II as transcription proceeds. Immediately upstream of the point of bond formation in RNA polymerase II, nascent RNA remains in hybrid with the DNA template for 8–9 bp. Structural information is available for the transcript in this region (12Gnatt A.L. Cramer P. Fu J. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1876-1882Crossref PubMed Scopus (746) Google Scholar, 13Westover K.D. Bushnell D.A. Kornberg R.D. Science. 2004; 303: 1014-1016Crossref PubMed Scopus (212) Google Scholar), but beyond the RNA-DNA hybrid, the location of the RNA within the transcription complex is unknown. A considerable segment of RNA remains inside the polymerase upstream of the point at which the transcript and template strand separate, since 17–19 nt of RNA within the transcription complex are protected from nuclease digestion and oligonucleotide hybridization probes (14Gu W.G. Wind M. Reines D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6935-6940Crossref PubMed Scopus (59) Google Scholar, 15Reeder T.C. Hawley D.K. Cell. 1996; 87: 767-777Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 16Újvári A. Pal M. Luse D.S. J. Biol. Chem. 2002; 277: 32527-32537Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Two possible exit paths have been proposed for the RNA (17Cramer P. Bushnell D.A. Fu J.H. Gnatt A.L. Maier-Davis B. Thompson N.E. Burgess R.R. Edwards A.M. David P.R. Kornberg R.D. Science. 2000; 288: 640-649Crossref PubMed Scopus (469) Google Scholar, 18Cramer P. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (968) Google Scholar). These models place the emerging transcript on opposing sides of the dock region of Rpb1, 1The abbreviations used are: Rpb1, Rpb2, etc., the largest, second largest, etc., subunits of RNA polymerase II; U2AF, U2 small nuclear ribonucleoprotein auxiliary splicing factor; U2AF65, the large subunit of U2AF; U2AF35, the small subunit of U2AF; RRM, RNA recognition motif; nt, nucleotide(s); CTD, C-terminal domain. the largest polymerase subunit. On path 1, the RNA passes around the base of the clamp domain toward subunit Rpb7, which contains both a ribonucleoprotein fold and an oligonucleotide binding fold (19Orlicky S.M. Tran P.T. Sayre M.H. Edwards A.M. J. Biol. Chem. 2001; 276: 10097-10102Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 20Todone F. Brick P. Werner F. Weinzierl R.O.J. Onesti S. Mol. Cell. 2001; 8: 1137-1143Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The Rpb4/Rpb7 heterodimer was shown to bind single-stranded DNA and RNA, and it has been suggested that Rpb7 may bind the emerging RNA transcript (10Armache K.-J. Kettenberger H. Cramer P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6964-6968Crossref PubMed Scopus (195) Google Scholar, 11Bushnell D.A. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6969-6973Crossref PubMed Scopus (222) Google Scholar, 13Westover K.D. Bushnell D.A. Kornberg R.D. Science. 2004; 303: 1014-1016Crossref PubMed Scopus (212) Google Scholar, 19Orlicky S.M. Tran P.T. Sayre M.H. Edwards A.M. J. Biol. Chem. 2001; 276: 10097-10102Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 20Todone F. Brick P. Werner F. Weinzierl R.O.J. Onesti S. Mol. Cell. 2001; 8: 1137-1143Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). On the alternative path, path 2, the RNA passes around the opposite face of RNA polymerase toward subunit Rpb8. Westover et al. (13Westover K.D. Bushnell D.A. Kornberg R.D. Science. 2004; 303: 1014-1016Crossref PubMed Scopus (212) Google Scholar) point out that in bacterial RNA polymerase only path 2 is electrostatically favorable. The simple extension of the RNA in their structure points toward this second groove (13Westover K.D. Bushnell D.A. Kornberg R.D. Science. 2004; 303: 1014-1016Crossref PubMed Scopus (212) Google Scholar). Several findings suggest that the transcript remains in contact with the polymerase for some time after emerging from within the active center. Our earlier results (16Újvári A. Pal M. Luse D.S. J. Biol. Chem. 2002; 277: 32527-32537Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) are consistent with a stabilizing interaction between the 5′-end of the RNA and the RNA polymerase at the point where the nascent transcript is 30–40 nt long. Hanna and Meares (21Hanna M.M. Meares C.F. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4238-4242Crossref PubMed Scopus (63) Google Scholar) showed that the 5′-end of transcripts generated with Escherichia coli RNA polymerase can cross-link to the polymerase up to a chain length of 94 nt. In order to better understand the exit path of the transcript from RNA polymerase II, we synthesized RNAs 17 nt and longer, which contained both cross-linkable residues and radioactive labels at their 5′-ends. We were surprised to discover that the emerging RNA immediately contacts U2AF65, a protein known to be involved both in splicing (reviewed in Ref. 22Moore M.J. Nat. Struct. Biol. 2004; 7: 14-16Crossref Scopus (78) Google Scholar) and in nuclear export (23Zolotukhin A.S. Tan W. Bear J. Smulevitch S. Felber B.K. J. Biol. Chem. 2002; 277: 3935-3942Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 24Blanchette M. Labourier E. Green R.E. Brenner S.E. Rio D.C. Mol. Cell. 2004; 14: 775-786Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). We show that the presence of U2AF65 can assist RNA polymerase in recovery into transcriptional competence at pause sites, and we suggest that the close association of U2AF65 and polymerase is important in facilitating the participation of U2AF65 in RNA processing events. Reagents—We obtained reagents from the following sources: fast protein liquid chromatography-purified NTPs from Amersham Biosciences, 32P-labeled NTPs from PerkinElmer Life Sciences, Bio-Gel A1.5m from Bio-Rad, oligo(dT)-cellulose and Deep Vent DNA polymerase from New England Biolabs, 5-iodo-UTP from Sigma, DNase I from Invitrogen, and streptavidin-coated paramagnetic beads from Promega. CpA dinucleotide was obtained as a custom synthesis from Dharmacon. DNA oligonucleotides were synthesized by Integrated DNA Technologies, Inc. The RNA oligonucleotides RNA2 and RNA3 used in Fig. 4C were synthesized with T7 RNA polymerase and subsequently purified on a 15% denaturing polyacrylamide gel. HeLa cells for nuclear extract preparation were purchased from the National Cell Culture Center. The 8WG16 antibody against RNA polymerase II was obtained from Abcam and the MC3 antibody against U2AF65 was a gift from Dr. Margarida Gama-Carvalho. Transcription Templates—Templates for transcription were all based on the adenovirus major late promoter. The construction of the pML20-42, pML20-40(6G), and pML20-40(17G) plasmids is described elsewhere (25Samkurashvili I. Luse D.S. Mol. Cell. Biol. 1998; 18: 5343-5354Crossref PubMed Scopus (44) Google Scholar, 26Pal M. Luse D.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5700-5705Crossref PubMed Scopus (27) Google Scholar). Plasmid pML220 was constructed by PCR amplifying a 120-bp segment of pGR220 (27Rice G.A. Kane C.M. Chamberlin M.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4245-4249Crossref PubMed Scopus (79) Google Scholar) with the following primers: 5′-CCGCTCGAGCACAAACGGCAAGATCGAGGG-3′ and 5′-CCCAAGCTTGCGGATAACAATTTCACACAGGAAACAGCTATGACC-3′. The PCR product was cut with restriction enzymes XhoI and HindIII and was cloned into pML20-42 between the XhoI and HindIII sites. This places the sequence CTTTTGTTCCCTTT 81 base pairs downstream from the transcription start site. To generate the pML222 plasmid, this sequence was altered by changing the underlined G to a T residue through the use of the Stratagene QuikChange™ site-directed mutagenesis kit. The DNA templates for transcription, which ranged in size from 190–298 bp, were produced by PCR. In all cases, the transcription start site was 96 bp from the upstream end of the fragment. For attached template experiments, the 5′ primer was biotinylated. DNA was purified using the Concert Rapid PCR Purification System (Marligen Bioscience) according to the manufacturer's instructions. Protein Purification—Recombinant human U2AF (consisting of U2AF35 and U2AF65) was expressed in and purified from baculovirus-infected High-Five cells (Invitrogen) as described previously (28Graveley B.R. Hertel K.J. Maniatis T. RNA. 2001; 7: 806-818Crossref PubMed Scopus (103) Google Scholar) with some modifications. The insect cells (0.74 g) were resuspended in 6 ml of lysis buffer (50 mm Tris, pH 7.9, 500 mm NaCl, 10% glycerol, 5 mm imidazole, 5 mm β-mercaptoethanol, and complete EDTA-free protease inhibitor mix, from Roche Applied Science) and sonicated two times for 10 s on ice. Nonidet P-40 was added to a final concentration of 1%. The lysate was cleared by centrifugation, and the supernatant was mixed with 0.5 ml of 50% Ni2+-nitrilotriacetic acid-agarose (equilibrated with lysis buffer) for 1 h at 4 °C. The resin was washed twice with 10 ml of lysis buffer plus 1% Nonidet P-40 and twice with 10 ml of wash buffer (50 mm Tris, pH 7.9, 500 mm NaCl, 20 mm imidazole, protease inhibitors, and 5 mm β-mercaptoethanol). The bound protein was eluted with two 1-ml aliquots of elution buffer (50 mm Tris, pH 7.9, 500 mm NaCl, 200 mm imidazole, protease inhibitors, and 5 mm β-mercaptoethanol) and was dialyzed overnight against BC100 (20 mm Hepes, pH 7.9, 100 mm KCl, 20% glycerol, 0.2 mm EDTA) plus 1 mm phenylmethylsulfonyl fluoride and 5 mm β-mercaptoethanol. In addition to the full-length U2AF65 and U2AF35, a 55-kDa fragment of U2AF65 was also visible by Coomassie staining after resolving the U2AF preparation on an SDS gel. Depletion of U2AF from Nuclear Extract—HeLa cell nuclear extract was depleted of U2AF by passage through an oligo(dT)-cellulose column at 1 m KCl as described by Valcarcel et al. (29Valcarcel J. Martinez C. Green M.R. Richter J.D. mRNA Formation and Function. Academic Press, Inc., New York1997: 31-53Crossref Google Scholar). A mock depletion reaction was carried out by making nuclear extract 1 m in KCl without exposure to oligo(dT)-cellulose, followed by dialysis against a 100 mm KCl buffer. Assembly and Purification of Ternary Transcription Complexes— Preinitiation complexes were assembled by incubating a mixture containing 50% HeLa nuclear extract and 14 μg/ml template DNA fragment at a final concentration of 75 mm KCl and 8 mm MgCl2 at 30 °C for 20 min, followed by gel filtration on Bio-Gel A1.5m to remove contaminating NTPs. The 1.9-ml gel filtration columns used BC100 (with 20 mm Tris-HCl, pH 7.9, instead of 20 mm Hepes) as the running buffer. Complexes U21 (pML20–40(6G) template) and U17 (pML20–40(17G) template) were generated by incubating preinitiation complexes with 1 mm CpA (initiating at position–1), 10 μm 5-iodo-UTP, 1 μm [α-32P]CTP, and 50 μm dATP as the energy source at 30 °C for 5 min. This produced initial transcripts of 6 nt (pML20–40(6G) template) or 7 nt (pML20–40(17G) template). These transcripts were then chased for 5 min at 30 °C by the addition of 200 μm CTP, UTP, and GTP (to generate U21 complexes) or 200 μm CTP, UTP, and ATP (to generate U17 complexes). The U21 and U17 complexes were then purified by the addition of Sarkosyl to 1% followed by gel filtration in MEM buffer (30 mm Tris-HCl, pH 7.9, 10 mm β-glycerophosphate, 62.5 mm KCl, 0.5 mm EDTA, and 1 mm dithiothreitol). This Sarkosyl rinsing procedure is described in detail in Ref. 30Izban M.G. Luse D.S. Genes Dev. 1991; 5: 683-696Crossref PubMed Scopus (199) Google Scholar. The transcripts in Sarkosyl-rinsed complexes were elongated by incubation for 5 min at room temperature with a 50 μm concentration of the appropriate NTPs as indicated in the figure legends. The experiment in Fig. 5 used templates attached to beads, which were assembled into preinitiation complexes as described (31Hawryluk P.J. Újvári A. Luse D.S. Nucleic Acids Res. 2004; 32: 1904-1916Crossref PubMed Scopus (22) Google Scholar). A23 complexes were generated by incubation with 100 μm ATP, 10 μm UTP, and 1 μm [α-32P]CTP at 30 °C for 5 min, followed by the addition of 10 μm CTP for an additional 5 min. After Sarkosyl rinsing, the A23 complexes were chased with all four NTPs at 30 °C as indicated in the legend to Fig. 5. Cross-linking—Samples were placed in microtiter plates in an ice water bath and irradiated for 20 min at 312 nm as described by Bartholomew et al. (32Bartholomew B. Dahmus M.E. Meares C.F. J. Biol. Chem. 1986; 261: 14226-14231Abstract Full Text PDF PubMed Google Scholar) using a Fisher Biotech transilluminator in the presence of aprotinin and leupeptin at 10 μg/ml. They were then treated with DNase I (0.3 units/μl) and, where indicated, RNase A (50 ng/μl) for 15 min at 37 °C. An aliquot from each sample was extracted with phenol and chloroform, ethanol-precipitated, and resolved on a 13% denaturing polyacrylamide gel to check the RNA content. The rest of the samples were dissolved in SDS loading buffer and resolved on 4–20% gradient polyacrylamide-SDS gels (Invitrogen). Gels were visualized on a STORM Imager (Amersham Biosciences). Immunoprecipitations—Cross-linked complexes were immunoprecipitated under nondenaturing conditions or after dissociation of the proteins under denaturing conditions. Complexes shown in Fig. 2 (left) were denatured in the presence of 1% SDS and 10 mm dithiothreitol by heating at 95 °C for 5 min and were subsequently diluted to a final concentration of 50 mm Hepes, pH 7.5, 150 mm NaCl, 1% Triton X-100, 0.1% SDS. Samples in the middle panel of Fig. 2 were in 50 mm Hepes, pH 7.5, 150 mm NaCl, and 1% Triton X-100, and samples on the right were in the buffer reported by Robert et al. (33Robert F. Blanchette M. Maes O. Chabot B. Coulombe B. J. Biol. Chem. 2002; 277: 9302-9306Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). All samples contained aprotinin and leupeptin at 10 μg/ml and were DNase I-digested (and RNase A-treated for samples in the right panel) before immunoprecipitation overnight at 4 °C with the 8WG16 anti-Rpb1 antibody (0.5 μg/ml) or MC3 anti-U2AF65 antibody (5 μl/ml of reaction). Gel Filtration—For the experiments in Fig. 4, A and B, 400-μl samples of transcription complexes were loaded onto a 40-ml (0.9 × 57-cm) gel filtration column (Bio-Gel A1.5m) that was equilibrated with MEM buffer. For Sarkosyl and salt rinsing, the column was preloaded with 400 μl of 400 mm KCl and 1% Sarkosyl in minimal essential medium before loading the sample that was also brought up to a final 400 mm KCl and 1% Sarkosyl. The excluded volume was collected in 750-μl fractions. Samples were supplemented with 8 mm MgCl2 and 10 μg/ml aprotinin and leupeptin. For the experiment in Fig. 4A, transcription complexes were advanced to U32 by the addition of 50 μm CTP, UTP, and GTP at room temperature for 5 min. Cross-linking took place as described above for 20 min. Samples were DNase I-digested (and, in A, RNase A-digested) before concentration by trichloroacetic acid precipitation. Structural studies have shown that the catalytic center of RNA polymerase II resides in a cleft formed by the two largest subunits, Rpb1 and Rpb2 (recently reviewed in Ref. 34Cramer P. Curr. Opin. Genet. Dev. 2004; 14: 218-226Crossref PubMed Scopus (68) Google Scholar). In order to investigate the path of the nascent RNA as it emerges from this central cleft, we have carried out RNA-protein cross-linking experiments in which UV cross-linkable residues were located near the 5′-end of the RNA. An earlier study (32Bartholomew B. Dahmus M.E. Meares C.F. J. Biol. Chem. 1986; 261: 14226-14231Abstract Full Text PDF PubMed Google Scholar) demonstrated UV cross-linking of 19–40-nt RNAs containing 4-thiouridine to the two largest subunits of RNA polymerase II. We found that whereas 4-thio-UTP was an acceptable substrate for extension of relatively long RNAs (∼150 nt) by RNA polymerase II, the polymerase would not incorporate 4-thio-UTP in the initial 5 nt of the transcript (data not shown). We therefore employed 5-iodo-UTP as our cross-linkable nucleotide. 5-Iodouridine shows specific photoreactivity with aromatic and sulfur-containing residues in proteins (35Meisenheimer K.M. Meisenheimer P.L. Koch T.H. Methods Enzymol. 2000; 318: 88-104Crossref PubMed Google Scholar). Cross-linking of the 5′ Region of the Transcript to Components of the Transcription Complex—The template employed in the initial phases of our study, called pML20–40(6G), is derived from the adenovirus major late promoter. The first 30 nt of the transcript from this template are shown in Fig. 1A. RNA polymerase II transcription complexes were assembled by incubating pML20–40(6G) DNA in HeLa nuclear extracts. Transcription was initiated with the dinucleotide primer CpA in the presence of low concentrations of 5-iodo-UTP and radioactive CTP. This resulted in RNA synthesis up to the G stop at position +5. After the addition of GTP and high concentrations of UTP and nonlabeled CTP, transcription proceeded to position +20. These complexes contained two 5-iodouridine residues and two neighboring radiolabeled cytidines at the 5′-end, but no cross-linkable or labeled residues were present beyond the sixth base (see Fig. 1A). We will use the convention of designating transcripts and the associated complexes by the length of the RNA and the final base incorporated; thus, pausing of CpA-primed transcription at +20 on pML20–40(6G) generates U21 complexes. U21 complexes were partially purified by Sarkosyl rinsing, which involves transient exposure to the detergent Sarkosyl during gel filtration (see "Materials and Methods"). As shown in Fig. 1B, transcripts in the Sarkosyl-rinsed U21 complexes could be extended very efficiently to generate A24, G26, or C28 complexes. Reactions containing U21, A24, G26, and C28 complexes were exposed to UV light, and the proteins that were labeled by cross-linking to the RNA were visualized after SDS-PAGE (Fig. 1C). Cross-linking of the U21 and A24 reactions resulted in the labeling of two major protein bands, of roughly 220–240 and 135 kDa (Fig. 1C, lanes 2 and 3). These are, as expected, the two largest subunits of RNA polymerase II, Rpb1 and Rpb2, based on their molecular weights and the reactivity of the upper band with anti-Rpb1 antibodies (Fig. 2) (see also Ref. 32Bartholomew B. Dahmus M.E. Meares C.F. J. Biol. Chem. 1986; 261: 14226-14231Abstract Full Text PDF PubMed Google Scholar). Reactions in which the transcript was extended only 2 or 4 bases further downstream, to G26 or C28, showed an additional band upon UV cross-linking (Fig. 1C, lanes 4 and 5). After RNase A treatment to truncate the cross-linked RNA, this protein displayed an apparent molecular mass of 65 kDa (Fig. 2, right). There was no significant cross-linking in control reactions, which were not exposed to UV (Fig. 1C, lane 1) or in which no 5-iodo-UTP was used (not shown). Identification of Proteins Cross-linked to the Transcript— Reactions containing U21 or C28 complexes were UV-cross-linked and then immunoprecipitated with the anti-Rpb1 antibody 8WG16 (Fig. 2). In control reactions, which were denatured before precipitation (lanes 1–4), only the largest of the three labeled bands was recovered, confirming that this protein is the largest subunit of RNA polymerase II. When the precipitations were done under native conditions, all three bands were recovered (lanes 5–8), indicating that the 65-kDa protein is part of the transcription complex and not simply cross-linked to free RNA that might have been released during the transcription reaction. We were surprised at the size of the 65-kDa cross-linked protein. We had expected from our detergent rinsing protocol that only core subunits of RNA polymerase II would be present in the transcript elongation complexes. However, no subunit of RNA polymerase II has a molecular mass close to the observed size. (Human Rpb2 and Rpb3 are 134 and 31 kDa, respectively; see Ref. 36McKune K. Moore P.A. Hull M.W. Woychik N.A. Mol. Cell. Biol. 1995; 15: 6895-6900Crossref PubMed Scopus (59) Google Scholar). We considered the possibility that, contrary to expectation, general transcription factors might remain in our complexes. The 62-kDa subunit of TFIIH and the largest subunit (58 kDa) of TFIIF have roughly the correct molecular weight, but we could find no evidence that either protein is responsible for the 65-kDa band (data not shown). A critical clue was provided by the observations of Robert et al. (33Robert F. Blanchette M. Maes O. Chabot B. Coulombe B. J. Biol. Chem. 2002; 277: 9302-9306Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), who showed that the splicing factor U2AF65 copurifies with RNA polymerase II through a TFIIS affinity column. The identity of the 65-kDa band was established by the fact that it could be recovered from cross-linking reactions using a monoclonal antibody (37Gama-Carvalho M. Krauss R.D. Chiang L. Valcarcel J. Green M.R. Carmo-Fonseca M. J. Cell Biol. 1997; 137: 975-987Crossref PubMed Scopus (111) Google Scholar) against U2AF65 (Fig. 2, lanes 9–12). U2AF65 (U2 small nuclear ribonucleoprotein auxiliary splicing factor, large subunit), together with U2AF35, forms the essential splicing factor U2AF (reviewed in Ref. 22Moore M.J. Nat. Struct. Biol. 2004; 7: 14-16Crossref Scopus (78) Google Scholar). U2AF65 recognizes the polypyrimidine tract of introns and recruits U2 small nuclear ribonucleoprotein to the branch point, whereas U2AF35 specifies the AG dinucleotide step at the 3′ splice site (22Moore M.J. Nat. Struct. Biol. 2004; 7: 14-16Crossref Scopus (78) Google Scholar, 38Zamore P.D. Patton J.G. Green M.R. Nature. 1992; 355: 609-614Crossref PubMed Scopus (458) Google Scholar). U2AF65 contains an N-terminal RS domain and three C-terminal RNA recognition motifs (RRM domains) (38Zamore P.D. Patton J.G. Green M.R. Nature. 1992; 355: 609-614Crossref PubMed Scopus (458) Google Scholar). Biochemical and structural studies suggest that only the first two RRM domains are involved in RNA recognition, and the third RRM represents a novel class of protein recognition motifs (39Kielkopf C.L. Lucke S. Green M.R. Genes Dev. 2004; 18: 1513-1526Crossref PubMed Scopus (194) Google Scholar). U2AF65 has also been shown to be involved in mRNA export (23Zolotukhin A.S. Tan W. Bear J. Smulevitch S. Felber B.K. J. Biol. Chem. 2002; 277: 3935-3942Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Recent studies on the Drosophila U2AF large subunit (U2AF50) implicate dU2AF50 in the expression and nuclear export of transcripts from both intron-containing and intronless genes (24Blanchette M. Labourier E. Green R.E. Brenner S.E. Rio D.C. Mol. Cell. 2004; 14: 775-786Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The minimal length of RNA which allows cross-linking to U2AF65 is informative. As noted above, about 17 bases of the nascent transcript are contained within RNA polymerase II, as judged by protection against both nuclease and oligonucleotide probes (14Gu W.G. Wind M. Reines D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6935-6940Crossref PubMed Scopus (59) Google Scholar, 15Reeder T.C. Hawley D.K. Cell. 1996; 87: 767-777Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 16Újvári A. Pal M. Luse D.S. J. Biol. Chem. 2002; 277: 32527-32537Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). U2AF65 protects 17 bases of a consensus RNA binding site from nuclease attack (see Ref. 40Kent O.A. Reayi A. Foong L. Chilibeck K.A. MacMillan A.M. J. Biol. Chem. 2003; 278: 50572-50577Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar and references therein); however, at another site, only a short polypyrimidine sequence of 6 nt is protected (41Gu H.D. Schoenberg D.R. Nucleic Acids Res. 2003; 31: 6264-6271Crossref PubMed Scopus (11) Google Scholar). Since U2AF65 recognizes its 17-nt site through two RRM domains (40Kent O.A. Reayi A. Foong L. Chilibeck K.A. MacMillan A.M. J. Biol. Chem. 2003; 278: 50572-50577Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), a single binding domain would recognize 8–9 nt, consistent with the protection seen at the nonconsensus site (41Gu H.D. Schoenberg D.R. Nucleic Acids Res. 2003; 31: 6264-6271Crossref PubMed Scopus (11) Google Scholar). Thus, our ability to detect cross-linking beginning with a 26-nt RNA suggests that U2AF65 binds to RNA immediately upon its emergence from within the RNA polymerase, probably through the use of a single RRM domain. Transcription in a U2AF-depleted Nuclear Extract—In order to investigate the role U2AF65 plays during transcription, U2AF65 and its a
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