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• W1984289421 abstract "ELL family transcription factors activate the rate of transcript elongation by suppressing transient pausing by RNA polymerase II at many sites along the DNA. ELL-associated factors 1 and 2 (EAF1 and EAF2) bind stably to ELL family members and act as strong positive regulators of their transcription activities. Orthologs of ELL and EAF have been identified in metazoa, but it has been unclear whether such RNA polymerase II elongation factors are utilized in lower eukaryotes. Using bioinformatic and biochemical approaches, we have identified a new Schizosaccharomyces pombe RNA polymerase II elongation factor that is composed of two subunits designated SpELL and SpEAF, which share weak sequence similarity with members of the metazoan ELL and EAF families. Like mammalian ELL-EAF, SpELL-SpEAF stimulates RNA polymerase II transcription elongation and pyrophosphorolysis. In addition, like many yeast RNA polymerase II elongation factors, deletion of the SpELL gene renders S. pombe sensitive to the drug 6-azauracil. Finally, phylogenetic analyses suggest that the SpELL and SpEAF proteins are evolutionarily conserved in many fungi but not in Saccharomyces cerevisiae. ELL family transcription factors activate the rate of transcript elongation by suppressing transient pausing by RNA polymerase II at many sites along the DNA. ELL-associated factors 1 and 2 (EAF1 and EAF2) bind stably to ELL family members and act as strong positive regulators of their transcription activities. Orthologs of ELL and EAF have been identified in metazoa, but it has been unclear whether such RNA polymerase II elongation factors are utilized in lower eukaryotes. Using bioinformatic and biochemical approaches, we have identified a new Schizosaccharomyces pombe RNA polymerase II elongation factor that is composed of two subunits designated SpELL and SpEAF, which share weak sequence similarity with members of the metazoan ELL and EAF families. Like mammalian ELL-EAF, SpELL-SpEAF stimulates RNA polymerase II transcription elongation and pyrophosphorolysis. In addition, like many yeast RNA polymerase II elongation factors, deletion of the SpELL gene renders S. pombe sensitive to the drug 6-azauracil. Finally, phylogenetic analyses suggest that the SpELL and SpEAF proteins are evolutionarily conserved in many fungi but not in Saccharomyces cerevisiae. The human ELL gene was originally identified as a gene that undergoes translocations with the trithorax-like MLL gene in acute myeloid leukemia (1Thirman M.J. Levitan D.A. Kobayashi H. Simon M.C. Rowley J.D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12110-12114Crossref PubMed Scopus (203) Google Scholar, 2Mitani K. Kanda Y. Ogawa S. Tanaka T. Inazawa J. Yazaki Y. Hirai H. Blood. 1995; 85: 2017-2024Crossref PubMed Google Scholar). Subsequently, ELL was purified from rat liver nuclear extracts based on its ability to activate the rate of transcript elongation by RNA polymerase II (pol II) 3The abbreviations used are: pol II, polymerase II; RACE, rapid amplification of cDNA ends; HPLC, high performance liquid chromatography; DTT, dithiothreitol; BSA, bovine serum albumin; ORF, open reading frame. in vitro. Mechanistic studies revealed that ELL interacts directly with transcribing pol II in vitro and functions by suppressing transient pausing by the enzyme at many sites along the DNA (3Shilatifard A. Lane W.S. Jackson K.W. Conaway R.C. Conaway J.W. Science. 1996; 271: 1873-1876Crossref PubMed Scopus (281) Google Scholar, 4Shilatifard A. Haque D. Conaway R.C. Conaway J.W. J. Biol. Chem. 1997; 272: 22355-22363Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Searches of sequence data bases identified two additional mammalian ELL family members, designated ELL2 and ELL3, as well as a single Drosophila melanogaster ortholog. All these proteins were shown to function similarly to activate the rate of elongation by pol II in vitro (5Shilatifard A. Duan D.R. Haque D. Florence C. Schubach W.H. Conaway J.W. Conaway R.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3639-3643Crossref PubMed Scopus (108) Google Scholar, 6Miller T. Williams K. Johnstone R.W. Shilatifard A. J. Biol. Chem. 2001; 275: 32052-32056Abstract Full Text Full Text PDF Scopus (61) Google Scholar, 7Gerber M. Ma J. Dean K. Eissenberg J.C. Shilatifard A. EMBO J. 2001; 20: 6104-6114Crossref PubMed Scopus (53) Google Scholar). EAF1 and EAF2 are two closely related proteins that were first identified in yeast two-hybrid screens for ELL-interacting proteins (8Simone F. Polak P.E. Kaberlein J.J. Luo R.T. Levitan D.A. Thirman M.J. Blood. 2001; 98: 201-209Crossref PubMed Scopus (64) Google Scholar, 9Simone F. Luo R.T. Polak P.E. Kaberlein J.J. Thirman M.J. Blood. 2003; 101: 2355-2362Crossref PubMed Scopus (67) Google Scholar). In mammalian cells, EAF1, EAF2, and ELL are colocalized in Cajal bodies, nuclear structures that are enriched in factors involved in transcription and mRNA processing (10Gall J.G. Bellini M. Wu Z. Murphy C. Mol. Biol. Cell. 1999; 10: 4385-4402Crossref PubMed Scopus (245) Google Scholar). Recently, we showed that EAF1 and EAF2 bind directly to ELL family members and function as strong positive regulators of ELL transcription activity in vitro (11Kong S.E. Banks C.A.S. Shilatifard A. Conaway J.W. Conaway R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10094-10098Crossref PubMed Scopus (71) Google Scholar). Although little is known about the role of the ELL-EAF complex in transcriptional regulation in vivo, the Drosophila gene encoding the ELL homolog Suppressor of Triplo-lethal (Su(Tpl)) is essential for viability in flies. Some mutations in Su(Tpl) suppress lethality resulting from overexpression of the Tpl gene, perhaps by impairing synthesis of Tpl mRNA (12Eissenberg J.C. Ma J. Gerber M.A. Christensen A. Kennison J.A. Shilatifard A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9894-9899Crossref PubMed Scopus (59) Google Scholar). The Xenopus laevis EAF2 protein functions during eye development to activate transcription of the gene encoding the essential Rx homeodomain transcription factor (13Maurus D. Hé;ligon C. Buörger-Schwaörzler A. Braöndlli A.W. Kuöhl M. EMBO J. 2005; 24: 1181-1191Crossref PubMed Scopus (90) Google Scholar). Consistent with a role for ELL in controlling transcript elongation in vivo, Drosophila ELL colocalizes with pol II at transcriptionally active sites on polytene chromosomes, and evidence suggests that mutations in Su(Tpl) may preferentially affect synthesis of some long transcripts (7Gerber M. Ma J. Dean K. Eissenberg J.C. Shilatifard A. EMBO J. 2001; 20: 6104-6114Crossref PubMed Scopus (53) Google Scholar). Many components of the pol II transcription machinery are highly conserved across species from mammals to yeast; however, until now attempts to identify orthologs of the ELL and EAF proteins in fungi have been unsuccessful, prompting speculation that elongation factors like ELL might have evolved only after the emergence of multicellular organisms, where genes can be many tens of kilobases long, and fine-tuning of the transcriptional program is expected to be particularly important for differentiation and development (12Eissenberg J.C. Ma J. Gerber M.A. Christensen A. Kennison J.A. Shilatifard A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9894-9899Crossref PubMed Scopus (59) Google Scholar). In this work, we identify in fungi genes that encode proteins similar to ELL and EAF. These genes are found in Schizosaccharomyces pombe and several other fungi with completely sequenced genomes but not in Saccharomyces cerevisiae. Like their counterparts from larger eukaryotes, S. pombe ELL and EAF (SpELL and SpEAF) interact with one another to form a stable heterodimer that potently activates transcription elongation by pol II in vitro. Like yeast strains bearing mutations in several other components of the pol II elongation machinery, S. pombe lacking the gene encoding ELL exhibit a 6-azauracil-sensitive phenotype, suggesting that ELL could play an important role in transcriptional regulation even in simple, unicellular organisms. Materials—Unlabeled ultrapure ribonucleoside 5′-triphosphates and [α-32P]CTP (400 mCi/mmol; 1 Ci = 37 GBq) were purchased from Amersham Biosciences. Recombinant RNasin ribonuclease inhibitor was obtained from Promega. Anti-FLAG (M2) monoclonal antibodies, anti-Myc (C-3956) rabbit polyclonal antibodies, anti-FLAG (M2)-agarose, and anti-FLAG peptide were purchased from Sigma. Light chain-specific anti-mouse antibodies were from Bethyl Laboratories and labeled with Alexa Fluor 680 (Invitrogen) according to the manufacturer’s instructions. Protein Sequence and Structure Analyses—Data base searches were performed using the PSI-BLAST program (14Altschul S.F. Madden T.L. Schaöffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar) with the standard parameters, i.e. profile inclusion cutoff 0.002, SEG filter, and composition-based statistics invoked; -h 0.002, -Ft, -T t. Multiple sequence alignments were done using the MUSCLE program (15Edgar R.C. BMC Bioinformatics. 2004; 5: 113Crossref PubMed Scopus (6100) Google Scholar). Fold recognition was done using the HHsearch program (16Soding J. Bioinformatics. 2005; 21: 951-960Crossref PubMed Scopus (1877) Google Scholar). Cloning of S. pombe ell1 and eaf1 Genes—The SMART™ RACE cDNA amplification system (Clontech) was used according to manufacturer’s instructions to identify the 5′ ends of each gene using the gene specific primers: 5′-GGGTGGAAGGCAAGGATTGCGGAGGAGA-3′ (ELL) or 5′-TGCTGGCCGTTTGGGATACTGTAGAGGG-3′ (EAF). RACE products were cloned into pCR®2.1-TOPO®, and the inserts were sequenced to identify the 5′ end of each gene and to confirm the positions of introns. Sequences corresponding to the coding regions of the mRNA were then subcloned into pBacPAK8 using primers overlapping consecutive exons to remove introns from S. pombe genomic DNA sequences. Strain Construction—The S. pombe strain used was PP138 h- (ade6-M216 leu1-32 ura4-D18 his3-D1). S. pombe was grown at 32 °C in rich medium (YES) supplemented with adenine, histidine, leucine, and uracil (225 μg/ml) or minimal medium (EMM) with the supplements indicated. The ell1Δ strain was generated by replacing the coding region of the ell1 gene with the kanMX6 marker as described (17Bahler J. Wu J.Q. Longtine M.S. Shah N.G. McKenzie A. II I Steever A.B. Wach A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 943-951Crossref PubMed Scopus (1771) Google Scholar). Analysis of Proteins Associated with FLAG-tagged SpELL and SpEAF in S. pombe—cDNAs encoding SpELL or SpEAF with N-terminal FLAG epitope tags were subcloned into a modified version of pNMT-TOPO (Invitrogen), which carries a thiamine repressible promoter, and transformed into S. pombe strain PP138. Cells were grown in EMM supplemented with 10 μm thiamine and 225 μg/ml each of adenine, histidine, and uracil. Once they had reached a density of ∼5 × 106/ml, cells were washed in EMM supplemented with adenine, histidine, and uracil alone to release thiamine repression and grown for an additional 18 h. Cultures were harvested by centrifugation at 3,000 × g, washed in cold H2O, and then washed in an extraction buffer containing 0.2 m Tris-HCl (pH 7.5), 0.39 m (NH4)2SO4, 10 mm MgSO4, 1 mm EDTA (pH 8.0), 20% v/v glycerol, 0.28 μg/ml leupeptin, 1.4 μg/ml pepstatin A, 0.17 mg/ml phenylmethylsulfonyl fluoride, and 0.33 mg/ml benzamidine. Cells were pulverized under liquid nitrogen by mortar and pestle, thawed, and resuspended in extraction buffer. Whole cell extracts were clarified by centrifugation at 150,000 × g before immunoprecipitation. FLAG-tagged SpELL or SpEAF and associated proteins were purified from whole cell extracts as described below under “Purification of Recombinant Proteins.” FLAG-immunopurified complexes were analyzed by MudPIT mass spectrometry as described previously (18Washburn M.P. Wolters D. Yates III J.R. Nat. Biotechnol. 2001; 19: 242-247Crossref PubMed Scopus (4099) Google Scholar, 19Wolters D. Washburn M.P. Yates J.R. Anal. Chem. 2001; 73: 5683-5690Crossref PubMed Scopus (1576) Google Scholar, 20Jin J. Cai Y. Yao T. Gottschalk A.J. Florens L. Swanson S.K. Gutierrez J.L. Coleman M.K. Workman J.L. Mushegian A. Washburn M.P. Conaway R.C. Conaway J.W. J. Biol. Chem. 2005; 280: 41207-41212Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 21Zybailov B. Mosley A.L. Sardiu M.E. Coleman M.K. Florens L. Washburn M.P. J. Proteome Res. 2006; 5: 2339-2347Crossref PubMed Scopus (827) Google Scholar). Expression of Recombinant Proteins in Insect Cells—cDNAs encoding wild type SpELL and SpEAF containing N-terminal FLAG or c-Myc epitope tags, as well as an SpEAF deletion mutant lacking the first 59 amino acids, were subcloned into pBacPAK8. Recombinant baculoviruses were generated with the BacPAK expression system (Clontech). Sf21 insect cells were cultured at 27 °C in Sf-900 II SFM (Invitrogen). Flasks containing 1 × 108 Sf21 cells were infected with the recombinant baculoviruses. Forty-eight hours after infection, cells were collected and lysed in 15 ml of ice-cold buffer containing 50 mm Hepes-NaOH (pH 7.9), 0.5 m NaCl, 5 mm MgCl2, 0.2% Triton X-100, 20%(v/v) glycerol, 0.28 μg/ml leupeptin, 1.4 μg/ml pepstatin A, 0.17 mg/ml phenylmethylsulfonyl fluoride, and 0.33 mg/ml benzamidine. Lysates were centrifuged 100,000 x g for 30 min at 4 °C. Purification of Recombinant Proteins—FLAG-tagged proteins were purified from Sf21 cell lysates by anti-FLAG agarose immunoaffinity chromatography. Lysates from 1 × 108 cells were incubated with 0.5 ml anti-FLAG (M2)-agarose beads overnight at 4 °C. The beads were washed three times with Tris-buffered saline (25 mm Tris-HCl (pH 7.4), 137 mm NaCl, 2.7 mm KCl), and bound proteins were eluted from the beads with Tris-buffered saline containing 10% (v/v) glycerol and 0.3 mg/ml FLAG peptide. Where indicated, anti-FLAG-agarose eluates prepared from Sf21 cells expressing both recombinant FLAG-SpELL and Myc-SpEAF were further purified by anion exchange HPLC. Eluates were adjusted to a conductivity equivalent to that of 0.05 m KCl and applied to a 0.6-ml TSK DEAE-NPR HPLC column (Tosoh-BioSep) equilibrated in Buffer A (40 mm Tris-HCl (pH 7.9), 1 mm EDTA, 1 mm DTT, 10% (v/v) glycerol) containing 0.1 m KCl. The column was eluted with a 6-ml linear gradient from 0.1 to 0.5 m KCl in Buffer A, and 0.2 ml fractions were collected. Concentrations of eluted proteins were estimated by using ImageQuant TL software (GE Healthcare) to compare the relative intensity of Coomassie Bluestained bands corresponding to full-length proteins to the intensity of bands corresponding to BSA standards after SDS-PAGE. Preparation of RNA Polymerase II—Mammalian pol II was purified from rat liver nuclei as described previously (22Conaway J.W. Conaway R.C. Science. 1990; 248: 1550-1553Crossref PubMed Scopus (55) Google Scholar), except that a TSK DEAE 5-PW HPLC column was used in place of TSK DEAE-NPR. Pol II from S. pombe was purified from a wild type strain (972h-) that had been grown to an OD600 of 8. The cells were dissolved at 1/2 volume/weight in 3× lysis buffer (300 mm Tris-HCl (pH 7.9), 450 mm KCl, 3 mm EDTA, 30 μm ZnCl2, 30 mm DTT, 15% glycerol, and 3× protease inhibitors) and lysed using continuous flow bead beating through a Dyno-Mill (Typ KDL, Willy A. Bachofen Maschinen Fabrik, Basel, Switzerland) connected to a coolflow system from Neslab (model HX-300). The lysate was cleared by centrifugation for 45 min at 8,000 rpm in a JLA 8.1 rotor (Beckman-Coulter). The supernatant was filtered through four layers of cheesecloth and applied to a heparin column equilibrated in HSB (50 mm Tris-HCl (pH 7.9), 1 mm EDTA, 10 μm ZnCl2, 10 mm DTT, 5% glycerol, 1× protease inhibitors) containing 150 mm KCl. The column was washed with 3 column volumes of the same buffer and then HSB containing 600 mm KCl. The eluate was precipitated with ammonium sulfate at 50% saturation, dissolved in TEZ (50 mm Tris-HCl (pH 7.5), 1 mm EDTA, 10 μm ZnCl2, 10 mm DTT, 1× protease inhibitors) until the conductivity of a 1:200 dilution was less that 100 μS/cm. The supernatant was clarified by centrifugation for 45 min at 35,000 rpm in a Ti-45 rotor and loaded onto an 8WG16 antibody-Sepharose column (CNBr-activated Sepharose coupled to monclonal antibody 8WG16 (23Thompson N.E. Aronson D.B. Burgess R.R. J. Biol. Chem. 1990; 265: 7069-7077Abstract Full Text PDF PubMed Google Scholar) at 2 mg/ml), which had been equilibrated with TEZ containing 500 mm ammonium sulfate. The column was equilibrated to room temperature and then washed with 25 column volumes of TEZ containing 500 mm ammonium sulfate. The protein was eluted with TEZ containing 500 mm ammonium sulfate and 50% glycerol. The eluate was diluted 5-fold with TEZ and applied to a Uno-Q1 column (Bio-Rad) that was equilibrated in TEZ with 100 mm ammonium sulfate. The column was washed with 5 column volumes of the same buffer and eluted with a 30 ml linear gradient from 100 mm ammonium sulfate to 600 mm ammonium sulfate in TEZ. Oligo(dC)-tailed Template Transcripton Assays—Oligo(dC) tailed pAd-GR220 was prepared as described (24Rice G.A. Kane C.M. Chamberlin M.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4245-4249Crossref PubMed Scopus (79) Google Scholar). 60-μl transcription assays were carried out in the presence of 20 mm Hepes-NaOH (pH 7.9), 20 mm Tris-HCl (pH 7.9), 60 mm KCl, 2 mm DTT, 0.5 mg/ml BSA, 3% (w/v) polyvinyl alcohol (average molecular mass 30,000-70,000 Da), 3% (v/v) glycerol, 8 units of RNasin, 8 mm MgCl2, ∼100 ng of oligo(dC)-tailed pAd-GR220, ∼25 ng of pol II, and ribonucleoside triphosphates and transcription factors as indicated in the figure legends. Reactions were preincubated for 5 min at 28 °C before the addition of ribonucleoside triphosphates. Reactions were stopped after incubation at 28 °C for the times indicated in the figures, and transcription products were resolved on 6% polyacrylamide gels containing 7 m urea, 45 mm Tris borate, and 1 mm EDTA (pH 8.3) and detected with a Molecular Dynamics Typhoon PhosphoImager. Preparation of Paused RNA Polymerase II Elongation Complexes—Paused pol II elongation complexes were assembled on oligo(dC) tailed pAd-GR220 by performing transcription reactions (scaled up 10-fold) in the presence of 50 μm ATP, 50 μm GTP, 2 μm CTP, and 10 μCi of [α-32P]CTP for 30 min at 28 °C. Free ribonucleoside triphosphates were removed by applying ∼600 μl of the reaction mixture to two sequential 4-ml Sephadex G-50 spin columns equilibrated in buffer containing 20 mm Hepes-NaOH (pH 7.9), 20 mm Tris-HCl (pH 7.9), 50 mm KCl, 1 mm DTT, 0.5 mg/ml BSA, 2% (w/v) polyvinyl alcohol, 3% (v/v) glycerol, and 5 mm MgCl2. The columns were spun for 5 min at 2,000 x g in a swinging bucket rotor, and 60 μl of eluant/reaction mixture was used in further experiments. Identification of ELL and EAF Orthologs in S. pombe—ELL and EAF homologs have not been characterized in the two best studied fungal model systems, S. cerevisiae and S. pombe.To evaluate the status of these proteins in fungi and other primitive eukaryotes, we searched for orthologs of ELL and EAF using the PSI-BLAST algorithm (14Altschul S.F. Madden T.L. Schaöffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar). When the full-length, 621-amino acid human ELL ORF was compared with the NCBI nonredundant protein data base, the only promising candidate for a yeast ELL ortholog was an S. pombe protein encoded by the predicted SPBP23A10.14c ORF, which appeared with a borderline E-value of 0.039 after the second iteration of the search. When SPBP23A10.14c itself was used as a query in PSI-BLAST searches, it nonetheless retrieved a predicted ortholog from the saccharomycete Yarrowia lipolytica (E-value 10-8), followed at the next iteration by predicted ELL orthologs from other fungi and higher eukaryotes (E-values between 10-4 and 10-8). Further iterations identified known and predicted ELL orthologs from all completely sequenced metazoa as well as from most protists and fungi but, notably, not from S. cerevisiae or from higher plants (Fig. 1A and data not shown). The predicted SPBP23A10.14c ORF encodes a 533-amino acid protein specified by a gene with 3 exons on S. pombe chromosome II. Sequencing of a cDNA generated by 5′-RACE confirmed the predicted SPB23A10.14c ORF. Multiple sequence alignments indicate that the sequence similarity between SPBP23A10.14c product and ELL orthologs from higher eukaryotes extends throughout the entire length of the protein, with the highest similarity concentrated in three predicted globular regions: an N-terminal all-β domain, a central region that includes a predicted all-α region, and the previously defined C-terminal occludin-like domain (Fig. 1A). We therefore designate the gene encoding the SPBP23A10.14c ORF as ell1, and we refer to the ell1 gene product as SpELL. When the full-length 268-amino acid human EAF1 ORF was compared with sequence databases, no highly scoring fungal homologs were found; however, using the highly conserved N-terminal 110 amino acids of the human EAF1 protein as the query, we detected a low scoring match to the predicted S. pombe SPCC1223.10c ORF (E-value of 7.8 at the second iteration). The predicted SPCC1223.10c ORF in the data base is 199 amino acids long and lacks 59 N-terminal amino acids that are highly conserved in metazoan EAF proteins. Using 5′-RACE we isolated from S. pombe total RNA a longer cDNA that included a new first exon and encoded 59 N-terminal amino acids missed in the original genome annotation (Fig. 1B). When this extended protein sequence was compared with the data base, the first match, which had a highly significant E-value of 4 × 10-7, was a protein from Schistozoma japonicum (gi 29841114) annotated as similar to Homo sapiens EAF1. When this S. japonicum sequence was used as a query, the known and predicted EAF homologs from various eukaryotes were detected. These included SpEAF, which joined the rest of the family at the fourth iteration of the search with an E-value of 10-4. EAF homologs were found in all metazoa, in higher plants, and in most fungi and protists but not in S. cerevisiae. Multiple sequence alignment and secondary structure prediction indicate that the EAFs consist of a non-conserved, highly charged, and most likely elongated or non-globular C-terminal domain and of a well conserved N-terminal domain that is predicted to adopt an all-β fold, broadly resembling an immuno-globulin-type β-sandwich, an arrangement commonly observed in domains mediating protein-protein interactions. We designate the gene encoding the extended SPCC1223.10c ORF as eaf1, and we refer to the eaf1 gene product as SpEAF. Yeast strains carrying mutations in genes encoding a number of proteins implicated in regulation of transcription elongation grow slowly in the presence of the nucleotide-depleting drug 6-azauracil. To determine whether ell1 or eaf1 mutants are sensitive to 6-azauracil, we generated strains lacking the ell1 or eaf1. ell1Δ and eaf1Δ strains were both viable. Although the eaf1Δ strain appeared to grow as well as wild type S. pombe on plates containing 6-azauracil, the ell1Δ strain exhibited a 6-azauracil-sensitive phenotype (Fig. 2 and data not shown). Direct Interaction of SpELL with SpEAF—To determine whether SpELL and SpEAF interact with one another in cells, N-terminal FLAG or V5 epitope-tagged SpELL or FLAG-tagged SpEAF were expressed individually in S. pombe under control of the inducible nmt1 promoter. SpELL- and SpEAF-associated proteins were purified from these strains using anti-FLAG or anti-V5 agarose immunoaffinity chromatography and identified by mass spectrometry using multi-dimensional protein identification technology (MudPIT). As summarized in Table 1, epitope-tagged SpELL and SpEAF specifically copurified with endogenous SpEAF and SpELL, respectively, indicating that the two proteins interact in cells.TABLE 1SpELL coimmunoprecipitates with SpEAF expressed in S. pombeFLAG-SpELLFLAG-SpEAFFLAG-controlPeptidesSpectraNSAFPeptidesSpectraNSAFPeptidesSpectraNSAFSpELL20700.0094361790.0123000SpEAF131170.0335322920.0425000 Open table in a new tab We next subcloned the SpELL and SpEAF ORFs into baculovirus vectors and expressed them in Sf21 insect cells in several epitope-tagged forms. In particular, we coexpressed FLAG-SpEAF with Myc-SpELL or FLAG-SpELL with Myc-SpEAF (Fig. 3, lanes 1-4) and purified the resulting protein complexes using anti-FLAG agarose chromatography (lanes 5-12). FLAG-SpEAF copurified with Myc-SpELL (lanes 5 and 9) and FLAG-SpELL copurified with Myc-SpEAF (lanes 7 and 11). The interaction of SpELL and SpEAF appeared to be direct, since only very small amounts of any additional proteins were present in our purified preparations of SpELL/SpEAF (lanes 9 and 11). The SpELL-SpEAF Complex Activates Transcription Elongation by S. pombe RNA Polymerase II—To test the effects of SpELL and SpEAF on transcription elongation by S. pombe pol II, we assayed transcript elongation on a linearized plasmid with a single-stranded 3′-oligo(dC)-tail on its template strand. Although purified pol II is unable to initiate from a specific location on double-stranded DNA without assistance from the general transcription factors TFIID, TFIIB, TFIIE, TFIIF, and TFIIH, it binds to the single-stranded oligo(dC)-tail and initiates transcription at the junction between the single- and double-stranded regions of the template (25Kadesch T.R. Chamberlin M.J. J. Biol. Chem. 1982; 257: 5286-5295Abstract Full Text PDF PubMed Google Scholar). On the template used in our experiments, the first nontemplate strand (dT) residue is 136 nucleotides downstream of the oligo(dC)-tail; thus, transcripts of 135 nucleotides will accumulate when transcription is initiated with just ATP, CTP, and GTP. In the experiment shown in Fig. 4, transcription was initiated by the addition of purified S. pombe pol II to reaction mixtures containing the oligo(dC)-tailed template, ATP, GTP, and [α-32P]CTP. After a 30-min incubation to allow accumulation of pol II ternary elongation complexes containing radioactively labeled, 135 nucleotide long transcripts, the elongation complexes were purified by gel filtration to remove unincorporated ribonucleoside triphosphates (Fig. 4, lane 1). Nascent transcripts were then chased into longer products by the addition of ATP, GTP, CTP and UTP, in the presence or absence of SpELL, SpEAF, or both (Fig. 4, lanes 2-13). Like the mammalian ELL-EAF complex (11Kong S.E. Banks C.A.S. Shilatifard A. Conaway J.W. Conaway R. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10094-10098Crossref PubMed Scopus (71) Google Scholar), the SpELL-SpEAF complex stimulated the rate of transcription elongation by its cognate S. pombe pol II, as detected by an increase in the rate that radioactively labeled, 135-nucleotide-long transcript was chased into longer products when reactions included the SpELL-SpEAF complex. Addition of either SpEAF or SpELL alone had a negligible effect on the rate of transcript elongation. Notably, SpEAF (60-251), which is the truncated version of SpEAF encoded by the predicted SPCC1223.10c ORF and which lacks the most highly evolutionarily conserved N-terminal region of the protein, was inactive even in the presence of SpELL, although SpEAF (60-251) could be coimmunoprecipitated with SpELL from lysates of insect cells coinfected with viruses encoding the two proteins (Fig. 5C).FIGURE 5Cochromatography of stimulatory activity with the SpELL-SpEAF complex. A, FLAG-SpELL and Myc-SpEAF were expressed separately (upper panel) or together (lower panel) in insect cells and purified by anti-FLAG-agarose chromatography. The eluate was adjusted to a conductivity equivalent to 0.05 m KCl and applied to a 0.6-ml TSK DEAE-NPR HPLC column pre-equilibrated in buffer A containing 0.1 m KCl. The column was eluted with a 6-ml linear gradient from 0.1 to 0.5 m KCl in buffer A, and 0.2-ml fractions were collected. Aliquots of the indicated fractions were analyzed by SDS-polyacrylamide gel electrophoresis and either stained with Coomassie Blue R-250 or analyzed by Western blotting with anti-FLAG (red) or anti-myc (green) antibodies. B, oligo(dC)-tailed template transcription reactions were performed in the presence of 50 μm ATP, 50 μm GTP, and 10 μCi [α-32P]CTP (400 mCi/mmol) with 3 pmol of SpELL, 3 pmol of SpEAF, 5 μg of α-amanitin, and 20 ng of pol II as indicated. Reactions were stopped after 5 min. C, upper panel, diagram of wild type SpEAF and SpEAF (60-251). The gray box represents the conserved globular domain predicted bioinformatically. Middle panel, oligo(dC)-tailed template assays were performed as described in B with SpELL and either wild type or mutant SpEAF; lower panel, FLAG-SpEAF (60-251) and Myc-SpELL were expressed in insect cells, immunoprecipitated with anti-FLAG antibodies, and analyzed by Western blotting as described in the legend to Fig. 3. D,5-μl aliquots of the indicated fractions from the TSK DEAE-NPR HPLC column shown in the lower panel of A were assayed for their ability to stimulate synthesis of 135-nucleotide transcripts. Transcription was initiated by the addition of 50 μm ATP, 50 μm GTP, and 10 μCi [α-32P]CTP (400 mCi/mmol) and stopped after a 4-min incubation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To obtain additional evidence that the SpELL-SpEAF complex possesses transcription activity, we further fractionated the purified SpELL-SpEAF complex by ion exchange HPLC and monitored copurification of SpELL-SpEAF with pol" @default.
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