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• W2036904418 abstract "We report the cDNA cloning and functional characterization of human cyclin L, a novel cyclin related to the C-type cyclins that are involved in regulation of RNA polymerase II (pol II) transcription. Cyclin L also contains a COOH-terminal dipeptide repeat of alternating arginines and serines, a hallmark of the SR family of splicing factors. We show that recombinant cyclin L interacts with p110 PITSLRE kinase, and that cyclin L antibody co-immunoprecipitates a kinase activity from HeLa nuclear extracts that phosphorylates the carboxyl-terminal domain (CTD) of pol II and splicing factor SC35, and is inhibited by the cdk inhibitor p21. Cyclin L antibody inhibits the second step of RNA splicing in vitro, and recombinant cyclin L protein stimulates splicing under suboptimal conditions. Significantly, the IC50 for splicing inhibition by p21 is similar to the IC50 for inhibition of the cyclin L-associated kinase activity. Cyclin L and its associated kinase are thus new members of the pre-mRNA processing machinery. We report the cDNA cloning and functional characterization of human cyclin L, a novel cyclin related to the C-type cyclins that are involved in regulation of RNA polymerase II (pol II) transcription. Cyclin L also contains a COOH-terminal dipeptide repeat of alternating arginines and serines, a hallmark of the SR family of splicing factors. We show that recombinant cyclin L interacts with p110 PITSLRE kinase, and that cyclin L antibody co-immunoprecipitates a kinase activity from HeLa nuclear extracts that phosphorylates the carboxyl-terminal domain (CTD) of pol II and splicing factor SC35, and is inhibited by the cdk inhibitor p21. Cyclin L antibody inhibits the second step of RNA splicing in vitro, and recombinant cyclin L protein stimulates splicing under suboptimal conditions. Significantly, the IC50 for splicing inhibition by p21 is similar to the IC50 for inhibition of the cyclin L-associated kinase activity. Cyclin L and its associated kinase are thus new members of the pre-mRNA processing machinery. cyclin-dependent kinase polymerase II glutathione S-transferase open reading frame monoclonal antibody Cyclins and their partners the cyclin-dependent kinases (cdks)1 (reviewed in Refs. 1Martı́n-Castellanos C. Moreno S. Trends Cell Biol. 1997; 7: 95-98Abstract Full Text PDF PubMed Scopus (51) Google Scholar and 2Pines J. Trends Biochem. Sci. 1993; 18: 195-197Abstract Full Text PDF PubMed Scopus (408) Google Scholar) may be classified into two major groups according to their function: the cell cycle regulators, which include the cyclin classes A, B, D, and E and cdks 1, 2, 3, 4, and the transcriptional regulators, comprising the cyclin classes C, H, K, and T and cdks 7, 8, and 9. These latter cyclin/kinase pairs are associated with the transcriptional machinery, and are components of transcription factor TFIIH (3Mäkelä T.P. Tassan J.P. Nigg E.A. Frutiger S. Hughes G.J. Weinberg R.A. Nature. 1994; 371: 254-257Crossref PubMed Scopus (234) Google Scholar, 4Roy R. Adamczewski J.P. Seroz T. Vermeulen W. Tassan J.P. Schaeffer L. Nigg E.A. Hoeijmakers J.H. Egly J.M. Cell. 1994; 79: 1093-1101Abstract Full Text PDF PubMed Scopus (390) Google Scholar, 5Fisher R.P. Morgan D.O. Cell. 1994; 78: 713-724Abstract Full Text PDF PubMed Scopus (560) Google Scholar, 6Cismowski M.J. Laff G.M. Solomon M.J. Reed S.I. Mol. Cell. Biol. 1995; 15: 2983-2992Crossref PubMed Scopus (189) Google Scholar, 7Feaver W.J. Svejstrup J.Q. Henry N.L. Kornberg R.D. Cell. 1994; 79: 1103-1109Abstract Full Text PDF PubMed Scopus (360) Google Scholar), elongation factor P-TEFb, (8Peng J. Zhu Y. Milton J. Price D. Genes Dev. 1998; 12: 755-762Crossref PubMed Scopus (452) Google Scholar, 9Napolitano G. Majello B. Licciardo P. Giordano A. Lania L. Gene (Amst.). 2000; 254: 139-145Crossref PubMed Scopus (43) Google Scholar, 10Fu T. Peng J. Lee G. Price D. Flores O. J. Biol. Chem. 1999; 274: 34527-34530Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), and the RNA polymerase II holoenzyme (11Rickert P. Seghezzi W. Shanahan F. Cho H. Lees E. Oncogene. 1996; 12: 2631-2640PubMed Google Scholar, 12Kuchin S. Yeghiayan P. Carlson M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4006-4010Crossref PubMed Scopus (161) Google Scholar, 13Liao S.M. Zhang J. Jeffrey D.A. Koleske A.J. Thompson C.M. Chao D.M. Viljoen M. van Vuuren H.J.J. Young R.A. Nature. 1995; 374: 193-196Crossref PubMed Scopus (367) Google Scholar). Human cyclin K is homologous to cyclin C, associates with the large subunit of pol II, and its kinase partner, cdk9, phosphorylates the CTD of pol II (14Edwards M.C. Wong C. Elledge S.J. Mol. Cell. Biol. 1998; 18: 4291-4300Crossref PubMed Scopus (78) Google Scholar). Phosphorylation of the CTD plays a pivotal role in regulating transcription initiation, elongation, and processing of RNA transcripts. It is widely accepted that transcription and RNA processing are linked (reviewed in Ref. 15Riedl T. Egly J. Gene Expr. 2000; 9: 3-13Crossref PubMed Scopus (47) Google Scholar): capping enzymes, polyadenylation factors, and splicing factors assemble at the CTD, and these interactions are modulated by CTD phosphorylation (reviewed in Refs. 16Corden J.L. Patturajan M. Trends Biochem. Sci. 1997; 22: 413-416Abstract Full Text PDF PubMed Scopus (149) Google Scholar, 17Neugebauer K.M. Roth M.B. Genes Dev. 1997; 11: 3279-3285Crossref PubMed Scopus (102) Google Scholar, 18Steinmetz E.J. Cell. 1997; 89: 491-494Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). Extensive research has focused on the role of the CTD in regulating pre-mRNA splicing. The CTD targets splicing factors to transcription sites in vivo (19Misteli T. Spector D.L. Mol. Cell. 1999; 3: 697-705Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar); phosphorylated pol II stimulates splicing (20Hirose Y. Tacke R. Manley J.L. Genes Dev. 1999; 13: 1234-1239Crossref PubMed Scopus (174) Google Scholar), and splicing factors associate with pol II through a hyperphosphorylated CTD (21Kim E., Du, L. Bregman D.B. Warren S.L. J. Cell Biol. 1997; 136: 19-28Crossref PubMed Scopus (213) Google Scholar). Splicing factors comprise the small nuclear ribonucleoprotein particles, the spliceosome-associated proteins, and the SR proteins (reviewed in Ref. 22Manley J.L. Tacke R. Genes Dev. 1996; 10: 1569-1579Crossref PubMed Scopus (605) Google Scholar). SR proteins constitute a conserved family of pre-mRNA splicing factors that are characterized by an arginine-serine dipeptide repeat within their carboxyl-terminal domain and one or two RNA-binding domains within their amino-terminal domain (23Graveley B. RNA. 2000; 6: 1197-1211Crossref PubMed Scopus (884) Google Scholar). SR proteins are essential splicing factors, and are capable of complementing splicing-deficient cellular extracts. Several members of this family have been identified, and among these the human factors ASF/SF2 (24Krainer A.R. Maniatis T. Ruskin B. Green M.R. Cell. 1984; 36: 993-1005Abstract Full Text PDF PubMed Scopus (427) Google Scholar, 25Krainer A.R. Mayeda A. Kozak D. Binns G. Cell. 1991; 66: 383-394Abstract Full Text PDF PubMed Scopus (413) Google Scholar) and SC35 (26Fu X.-D. Maniatis T. Science. 1992; 256: 535-538Crossref PubMed Scopus (192) Google Scholar) are well characterized. Four additional SR proteins with molecular masses of 30, 40, 55, and 75 kDa have also been identified (27Roth M.B. Zahler A.M. Stolk J.A. J. Cell Biol. 1991; 115: 587-596Crossref PubMed Scopus (267) Google Scholar). SR proteins are required for early steps in spliceosome assembly and influence selection of splice sites (22Manley J.L. Tacke R. Genes Dev. 1996; 10: 1569-1579Crossref PubMed Scopus (605) Google Scholar, 26Fu X.-D. Maniatis T. Science. 1992; 256: 535-538Crossref PubMed Scopus (192) Google Scholar,28Krainer A.R. Conway G.C. Kozak D. Cell. 1990; 62: 35-42Abstract Full Text PDF PubMed Scopus (336) Google Scholar). In contrast, the SR-related proteins contain an RS repeat but lack RNA-binding domains (reviewed in Ref. 29Blencowe B. Bowman J. McCracken S. Rosonina E. Biochem. Cell Biol. 1999; 77: 277-291Crossref PubMed Scopus (107) Google Scholar). A recent survey of the human, yeast, Drosophila melanogaster, andCaenorhabditis elegans genomes has identified a number of other RS domain proteins. These include proteins involved in 3′-end processing, chromatin-associated proteins, kinases, phosphatases, and a new cyclin, named cyclin L, that was identified in C. elegans and D. melanogaster (30Boucher L. Ouzounis C.A. Enright A.J. Blencowe B.J. RNA. 2001; 7: 1693-1701PubMed Google Scholar), and it is similar to cyclin ania-6 in the mouse (31Berke J.D. Sgambato V. Zhu P.-P. Lavoie B. Vincent M. Krause M. Hyman S.E. Neuron. 2001; 32: 277-287Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Cyclin L is related to the transcriptional cyclin K (14Edwards M.C. Wong C. Elledge S.J. Mol. Cell. Biol. 1998; 18: 4291-4300Crossref PubMed Scopus (78) Google Scholar), and it is the first known example of a cyclin containing an RS domain in addition to a cyclin box. The function of cyclin L is not known although results obtained from immunofluorescence and immunoprecipitation experiments show that the mouse homologue, cyclin ania-6a, localizes to nuclear speckle compartments, associates with the hyperphosphorylated form of RNA pol II, the splicing factor SC-35, and the cdk PITSLRE (31Berke J.D. Sgambato V. Zhu P.-P. Lavoie B. Vincent M. Krause M. Hyman S.E. Neuron. 2001; 32: 277-287Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). These results suggest a potential role for cyclin L in RNA splicing. We report here the cloning and characterization of the human gene for cyclin L, and show that recombinant human cyclin L interacts with p110 PITSLRE kinase. Moreover, cyclin L is associated with a kinase activity that phosphorylates histone H1, the CTD, and SR protein SC35. This activity is inhibited by low concentrations of the cdk-specific inhibitor p21. An antibody to cyclin L inhibits in vitrosplicing specifically at the second step, and recombinant cyclin L protein stimulates splicing of a β-globin precursor RNA. Furthermore,in vitro splicing is inhibited by p21 with an inhibition profile nearly identical to that of kinase inhibition. These results provide initial evidence that cyclin L is a functional cyclin and directly affects pre-mRNA splicing, although the precise mechanism remains to be elucidated. Humancyclin L cDNA was generated by PCR using primers designed to match the 5′- and 3′-ends of a contiguous sequence of expressed sequence tags that appeared to encode a novel cyclin. The primers were: sense, 5′-CAGTCTTGTTTCGGGTTCCGGCTGCGTT-3′ and antisense, 5′-AAAAACAAGATTTGTATTTTATTTCCTTGT-3′. These primers were used to amplify the cDNA clone from human lung cDNA (CLONTECH) using a thermostable polymerase mixture (Advantage cDNA polymerase, CLONTECH). The PCR product was excised from a low-melting point agarose gel, and the agarose digested with AgarAce (Promega), cloned into the T-A vector pCR-II-TOPO (Invitrogen, The Netherlands), and sequenced in both directions using an Applied Biosystems 373A sequencer. A membrane containing 2 μg of poly(A)+ RNA from eight different human tissues (CLONTECH) was probed with a 738-bp PCR fragment spanning positions 111–849 of the cyclin L cDNA. 20 ng of the PCR fragment was labeled using the High Prime DNA Labeling kit (Roche Molecular Biochemicals) and purified using a Chromaspin-30 column (CLONTECH). The hybridization, stripping, and re-hybridization procedures were as described by the manufacturer, except that the last wash was carried out at 42 °C. The coding sequence of thecyclin L cDNA clone described above was amplified using primers containing an overhanging EcoRI recognition site (upstream primer, 5′-CGTCGGAATTCACGCGTCCGGGCCTCATTCG-3′ and downstream primer, 5′-GTCGGAATTCGGCGCCTGTGCCTGCCATGTC-3′). The PCR product was purified, digested with EcoRI, and cloned into the EcoRI site of pGex2T (Amersham Biosciences). A plasmid containing the correct orientation of the insert as confirmed by sequencing was used for protein expression. Cultures of freshly transformed XL1 Blue cells (Stratagene) were grown to anA 600 of 1.0, induced with 1 mmisopropyl-1-thio-β-d-galactopyranoside, incubated for an additional 2 hours, and harvested by centrifugation. Cells were lysed for 45 min in ice-cold TBSE (10 mm Tris-Cl, pH 8.0, 150 mm NaCl, 2 mm EDTA) containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 0.5 μg/ml pepstatin) and lysozyme (0.2 mg/ml). The crude lysate was sonicated, and Triton X-100 was added to a final concentration of 1% (v/v). Cell debris was removed by centrifugation at 12,000 × g for 30 min. The supernatant was incubated for 30 min at 4 °C with washed glutathione-Sepharose beads, followed by three washes with phosphate-buffered saline, and eluted in 10 mm glutathione in 50 mm Tris-Cl, pH 8.0, 150 mm NaCl, 1% phenylmethylsulfonyl fluoride. Proteins were dialyzed in phosphate-buffered saline + 0.1% phenylmethylsulfonyl fluoride using Slide-A-Lyzer cassettes (Pierce). For GST interaction experiments, 4 μg of either GST-cyclin L or GST alone was added to 10-μl aliquots of HeLa nuclear extract (containing 80 μg of total protein) in a total volume of 100 μl in a buffer containing 10 mm Tris-Cl, pH 7.6, 100 mm NaCl, and 1 mm dithiothreitol. After incubation at 30 °C for 1 h, these samples were then incubated with 20 μl of packed glutathione-Sepharose beads (Amersham Biosciences) on a rocker platform at 4 °C for 2 h. The beads were prewashed with the buffer listed above containing 500 μg/ml bovine serum albumin (three washes with 10 volumes each). After incubation, the beads were pelleted and then washed five to six times with 500 μl of buffer containing 0.1% Nonidet P-40. This extensive washing was necessary to reduce nonspecific binding. The washed beads were either used directly for kinase assays, or bound proteins were eluted in SDS sample buffer (at 95 °C for 5 min) and subjected to SDS-PAGE and Western blotting (see below). The mouse anti-SR protein (1H4, also known as 1H4G7) monoclonal antibody was from Zymed Laboratories Inc. Anti-PITSLRE (C-17), anti-cyclin C (T-19), anti-RB, anti-CBP, anti-pol II (N-20), anti-TFIIH (p89), and anti-cdk6 were purchased from Santa Cruz Biotechnology, Inc. Antibody to recombinant cyclin L (anti-rL) was raised in rabbits against gel-purified GST-cyclin L fusion protein p36 (see below). Purified GST-cyclin L was separated by a preparative SDS-polyacrylamide gel alongside a prestained molecular weight marker and blotted onto nitrocellulose. The regions containing the p36 or p70 peptide were cut out and blocked in Blotto (5% dry milk, 160 mm NaCl, 20 mmNaH2PO4 buffer, pH 7.2, and 0.1% Tween 20) for 1 h at ambient temperature. Serum containing anti-L was incubated separately with each nitrocellulose strip at 4 °C overnight, and the blots were washed for 20 min in 0.15 m NaCl followed by a 20-min wash in phosphate-buffered saline. Bound antibody from each strip was eluted in 400 μl of glycine, pH 2.8, followed by immediate neutralization in 0.1 m Tris-Cl, pH 8.3. SDS gels were transferred to Magnagraph nylon membranes in 191 mm glycine, 25 mm Tris base, and 20% methanol using a semi-dry blotting apparatus. Membranes were blocked in Blotto (5% dry milk, 160 mm NaCl, 20 mm NaH2PO4 buffer, pH 7.2, and 0.1% Tween 20) overnight at 4 °C. At the same time, the anti-cyclin L antibody was incubated in Blotto (1:2000) overnight to reduce nonspecific binding. Filters were washed in TBST (50 mmTris-Cl, pH 7.5, 150 mm NaCl, 0.1% Tween 20) and incubated for 1 h in antibody/Blotto solution. After washing three times for 10 min in TBST, the filter was incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (H+L) (Bio-Rad) (1:2000). After extensive washing of the membranes, signals were detected using the SuperSignal West Pico chemiluminescent substrate (Pierce). Protein A-Sepharose was washed three times in A100 buffer (20 mm Hepes-OH, pH 7.2, 100 mm KCl, 0.1 mm EDTA, 0.2 mm dithiothreitol) and incubated overnight at 4 °C with 10 μl of bovine serum albumin (1.5 mg/ml) and 10 μl of antibody (as indicated). The complex was washed as before, and aliquots (1/5 volume) were incubated with 80 μg of HeLa nuclear extract for 1 h at 4 °C. The complexes were washed as before with A100 + 0.1% Nonidet P-40 followed by three washes with A100. The pellets were resuspended in A100 to give a total volume of 120 μl. 60 μl each were added to 10 μl of a kinase reaction containing 20 μm ATP, 1 μl of [α-32P]ATP (150 μCi/μl), 6 mmMgCl2, and the kinase substrate as indicated. The reaction was incubated at ambient temperature for 1 h, precipitated with 25% (v/v) trichloroacetic acid, washed with acetone, and air-dried. Pellets were resuspended in SDS-loading buffer, boiled, and separated by SDS-PAGE. Gels were dried and exposed to Bio-Max film or quantified on a PhosphorImager (Molecular Dynamics). The precursor RNA used in in vitro splicing assays was transcribed from plasmid SP64Hβδ6 containing a β-globin precursor coding sequence (24Krainer A.R. Maniatis T. Ruskin B. Green M.R. Cell. 1984; 36: 993-1005Abstract Full Text PDF PubMed Scopus (427) Google Scholar). The plasmid was digested to completion with BamHI, phenol/chloroform extracted, and used in a transcription reaction with SP6 RNA polymerase in the presence of 7-methyl-G cap analog and [α-32P]UTP as described by the manufacturer (Promega). The labeled RNA was run on a 6% denaturing polyacrylamide gel, the gel was briefly exposed to film, and the band corresponding to the full-length RNA was cut from the gel. RNA was eluted from the gel slice in elution buffer (0.3m Na-acetate, pH 5.3, 33% buffer-saturated phenol, 60 μg of glycogen) for 3 h at ambient temperature or overnight at 4 °C (32Yuryev A. Patturajan M. Litingtung Y. Joshi R.V. Gentile C. Gebara M. Corden J.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6975-6980Crossref PubMed Scopus (289) Google Scholar). The sample was centrifuged, and the supernatant was purified through an Ultrafree CL filter (Millipore), aliquoted, precipitated separately with ethanol, and stored as dry pellets at −80 °C. Pellets were resuspended in RNase-free H2O at an estimated concentration of 6 ng/μl (or 40 fmol/μl) and 1–2 μl were used in in vitro splicing reactions. Splicing reactions were performed as described (33Reichert V. Moore M. Nucleic Acids Res. 2000; 28: 416-423Crossref PubMed Scopus (23) Google Scholar) and contained 80 mmpotassium acetate, 4 mm magnesium acetate, 20 mm creatine phosphate, 1 mm freshly prepared ATP, 1 unit/ml RNasin (Promega), labeled β-globin RNA precursor, 40 μg of HeLa nuclear extract (Promega), or 20 μg HeLa nuclear extract plus 200 ng of GST-cyclin L fusion protein, in a total volume of 20 μl. Reactions were incubated at 30 °C for 3 h (or as indicated), stopped with 180 μl of stop buffer (0.5% SDS, 0.3m Na-acetate, pH 5.3, in TE) plus 20 μg of glycogen, extracted with phenol/chloroform, and precipitated with ethanol. The reaction products were run on 6% denaturing polyacrylamide gels with a kinase-labeled 100-bp ladder (PerkinElmer Life Sciences), and were visualized by exposure to Bio-Max film and phosphorimage analysis. A 2076-bp human cyclin LcDNA was cloned by PCR from a human lung cDNA library (GenBankTM accession number AF180920). The first methionine codon, at nucleotides 55–57, is in a strong sequence context for an initiation consensus sequence (34Kozak M. Nucleic Acids Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4172) Google Scholar), suggesting that this is the correct initiation codon. The ORF encodes a theoretical 59.6-kDa protein, 526 amino acid residues in length, with an isoelectric point of 10.71. A classical polyadenylation signal is located at nucleotide positions 2054–2059. BLAST searches with the predicted amino acid sequence revealed a cyclin box spanning amino acids 48–255 that shares 32% identity and 52% similarity with the cyclin box of cyclin K (14Edwards M.C. Wong C. Elledge S.J. Mol. Cell. Biol. 1998; 18: 4291-4300Crossref PubMed Scopus (78) Google Scholar) (Fig.1 A). BLAST searches with smaller portions of the predicted amino acid sequence identified a region rich in serine-arginine repeats at the COOH terminus that is highly similar to the RS repeats that are conserved in all the members of the SR protein family of mRNA splicing factors. An alignment of the RS repeats of cyclin L and the human alternative splicing factor ASF/SF2 (25Krainer A.R. Mayeda A. Kozak D. Binns G. Cell. 1991; 66: 383-394Abstract Full Text PDF PubMed Scopus (413) Google Scholar) is shown in Fig. 1 B. A region of 42 amino acids is 77% similar and 66% identical in these proteins, and the location of the RS repeat at the COOH terminus is conserved in both proteins. No homologies to known RNA-binding domains were identified in cyclin L. The tissue distribution and size of cyclin L mRNA was analyzed by Northern blot with poly(A)+ RNA from different human tissues, probed with a radiolabeled 700-bp DNA fragment from the 5′-region of the cyclin L cDNA (Fig. 1 C). Two major bands of 2.3 and 4.5 kb are detected in all the tissues examined and represent alternatively spliced products, as discussed below. The 4.5-kb band is weaker than the 2.3-kb band in most tissues, except in thymus and lymphocytes that express high levels of the 4.5-kb RNA. Identical results were obtained with a DNA probe derived from the 3′ end of the cyclin L cDNA, confirming the authenticity of this cDNA. The human cyclin L gene is located on chromosome 3 in the 3q23.2–3 region. The cyclin L gene is 12.4 kb in length, and is encoded by 14 exons (Fig.2 A) on a 500,000-bp scaffold segment from 156227351 to 156727350 (Celera accession number GA_x54KRCCA4FB) (35Venter J. Adams M. Myers E., Li, P. Mural R. Sutton G. Smith H. Yandell M. Evans C. Holt R. et al.Science. 2001; 291: 1304-1351Crossref PubMed Scopus (10698) Google Scholar). There is a CpG island (67% GC) that encompasses the whole of exon 1 and most of exon 2. A TATA-less promoter is predicted by the Gene-Finder program (36Solovyev V. Salamov A. Gaasterland T. Karp P. Karplus K. Ouzounis C. Sander C. Valencia A. Proceedings of the Fifth International Conference on Intelligent Systems for Molecular Biology. AAAI Press, Menlo Park, CA1997: 294-302Google Scholar), with the transcription start site located 89 bp upstream of the initiation codon. A comparison of the human and mouse cyclin L genes (GenBankTMaccession number AF185590) shows them to be very similar in organization (data not shown). Both genes are ∼12 kb in length, with all shared internal exons of identical length. Exon 8 does not appear to be a coding exon as it was only found in the 3′-untranslated region of the β transcript (see below), and the equivalent of exon 8 is absent from the mouse gene. cDNA cloning revealed that the human cyclin L gene generates a number of alternatively spliced mRNA transcripts. The major transcript has been designated the α transcript that encodes a 526-amino acid residue ORF (Fig.2 B). Exons 4, 7, and 8 are skipped in cyclin Lα mRNA. Cyclin Lα mRNA has 92% identity with the mouse cyclin ania-6a ORF (GenBankTM accession number AF159159) (31Berke J.D. Sgambato V. Zhu P.-P. Lavoie B. Vincent M. Krause M. Hyman S.E. Neuron. 2001; 32: 277-287Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The only region of divergence between humancyclin Lα and mouse ania-6a is close to the NH2 terminus in a repetitive region. Two other human transcripts encode truncated proteins. The β transcript skips exon 4, terminates in exon 7, and encodes a 232-amino acid residue ORF (GenBankTM accession number AF367476). The γ transcript encodes a 172-amino acid residue ORF, includes exon 4, and terminates within this exon (GenBankTM accession number AY034790). Both the β and γ transcripts are conserved in mouse, where it was shown that the truncated variant is not targeted to the nucleus and does not associate with RNA pol II and splicing factors (31Berke J.D. Sgambato V. Zhu P.-P. Lavoie B. Vincent M. Krause M. Hyman S.E. Neuron. 2001; 32: 277-287Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). As noted above, there are 2 major cyclin L transcripts of 2.3 and 4.5 kb detected on a Northern blot (Fig. 1 C). The 2.3-kb band likely represents the α and β transcripts, and an analysis of human and mouse expressed sequence tag data suggests that the 4.5-kb band may represent the γ transcript. Cyclin Lγ is generated by read-through of the donor splice sites from exons 5 to the polyadenylation signal. A cDNA for cyclin Lγ was obtained by screening a human lymphocyte cDNA library. Consistent with this finding, peripheral blood lymphocytes express a large amount of the 4.5-kb cyclin Lγ mRNA (Fig. 1 C). In an effort to clone the human homologue for the B“ subunit of the yeast transcription factor TFIIIB, we used a rabbit polyclonal antibody raised against the yeast protein (kindly provided by Drs. E. P. Geiduschek and G. A. Kassavetis, University of California, San Diego) to screen a human lymphocyte expression cDNA library. Surprisingly, all the clones obtained from this screen were identified as cyclin Lγ, suggesting that this antibody recognizes one or more epitopes in the human cyclin L protein. We therefore refer to this antibody as anti-L. Moreover, this antibody recognizes a single major band of ∼55–60 kDa in HeLa nuclear extracts (Fig.3 B, lane 3) (the size appears to fluctuate slightly depending on the experiment, probably due to different phosphorylation levels of cyclin L). To determine that this band in HeLa nuclear extracts is cyclin L, we expressed the cyclin Lα cDNA as a GST fusion protein in bacteria and affinity purified the antibody against the recombinant cyclin L protein. Expression of GST-cyclin L in bacteria yields two major bands that migrate at ∼36 and 70 kDa (referred to as p36 and p70). These proteins were identified as cyclin L, since they are both recognized by anti-cyclin L antibody (Fig. 3 A). It was concluded that p36 is a truncated version of the GST-cyclin L protein and not a degradation product, since the band was expressed even in the presence of a protease inhibitor mixture. p70 likely represents the full-length fusion protein, although it migrates slightly faster on SDS gels than the predicted molecular mass. Anti-L antibody was affinity-purified using nitrocellulose filter strips that either contained the GST-cyclin L p36 or the p70 peptide. Purified anti-L antibody was then used to probe HeLa nuclear extract, and it recognizes the same 55–60-kDa band as anti-L serum (Fig. 3 B,lane 3) regardless of whether it was purified against p70 or p36 (lanes 1 and 2, respectively). This experiment shows that anti-L recognizes a protein in HeLa nuclear extract that is identical to recombinant cyclin L. Furthermore, a polyclonal antibody raised against recombinant GST-cyclin L (anti-rL) also recognizes the same 55–60-kDa protein in HeLa nuclear extract (data not shown). Anti-L antibody was used to immunoprecipitate cyclin L and any potential kinase partner(s) from a HeLa nuclear extract. Immunoprecipitates were bound to Protein A-Sepharose, washed extensively, and used in an in vitro kinase assay. The substrate specificity of the cyclin L-associated kinase was analyzed with recombinant RNA pol II CTD (GST-CTD), histone H1, splicing factors SC35 and SRp46, GST-cyclin L fusion protein as test substrates, and bovine serum albumin as a negative control. The cyclin L-associated kinase specifically phosphorylates the CTD of pol II, histone H1, and SC35 (Fig. 4 A). In contrast, SRp46, GST-cyclin L, and bovine serum albumin are not substrates for the cyclin L-associated kinase. The lack of phosphorylation of cyclin L and SRp46 demonstrates that the cyclin L-associated kinase is not a general SR protein kinase. As an additional control, these same substrates were tested with immunoprecipitates formed with preimmune serum, and as expected, no kinase activity was detected (Fig.4 A, lanes labeled PI). To confirm that the kinase activity was associated with cyclin L, and was not an artifact of the immunoprecipitation procedure, we incubated purified GST-cyclin L protein with HeLa extract, captured GST-cyclin L, and associated proteins on glutathione-Sepharose beads and used the washed beads in an in vitro kinase assay. A potent kinase associates with GST-cyclin L, but not with GST alone as evidenced by the phosphorylation of histone H1 (Fig. 4 B). In additional experiments, we compared the kinase activity of immunoprecipitates formed with anti-cyclin L with those isolated with other antibodies. We used anti-TFIIIA as negative control, and antibodies to the p62 subunit of TFIIH for cdk7 activity, and anti-cdk8 as positive controls for CTD phosphorylation (Fig. 4 C). The CTD kinase activity of the anti-cyclin L IP is comparable with that of anti-p62 and anti-cdk8. Anti-cyclin L and anti-p62 IPs also phosphorylate histone H1, whereas anti-cdk8 immunoprecipitates failed to phosphorylate H1 as expected (11Rickert P. Seghezzi W. Shanahan F. Cho H. Lees E. Oncogene. 1996; 12: 2631-2640PubMed Google Scholar). No significant levels of kinase activity were detected with the preimmune serum and no antibody control reactions. To provide an independent confirmation that a kinase activity is associated with cyclin L, we partially purified cyclin L and associated proteins from HeLa nuclear extracts through three successive rounds of ion exchange chromatography. Fractions containing cyclin L were identified by immunoblotting with anti-cyclin L antibody. Following phosphocellulose, DEAE-Sepharose, and Mono-Q FPLC, fractions that contained cyclin L protein al" @default.
• W2036904418 created "2016-06-24" @default.
• W2036904418 creator A5056757448 @default.
• W2036904418 creator A5060525515 @default.
• W2036904418 creator A5085467911 @default.
• W2036904418 creator A5088908077 @default.
• W2036904418 date "2002-07-01" @default.
• W2036904418 modified "2023-10-14" @default.
• W2036904418 title "Cyclin L Is an RS Domain Protein Involved in Pre-mRNA Splicing" @default.
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