Conditional Expression of RNA Polymerase II in Mammalian Cells
2000; Elsevier BV; Volume: 275; Issue: 32 Linguagem: Inglês
10.1074/jbc.m001883200
ISSN1083-351X
AutoresMark Meininghaus, Rob D. Chapman, Manuela Horndasch, Dirk Eick,
Tópico(s)DNA Repair Mechanisms
ResumoThe carboxyl-terminal domain (CTD) of the large subunit of mammalian RNA polymerase II contains 52 repeats of a heptapeptide that is the target of a variety of kinases. The hyperphosphorylated CTD recruits important factors for mRNA capping, splicing, and 3′-processing. The role of the CTD for the transcription process in vivo, however, is not yet clear. We have conditionally expressed an α-amanitin-resistant large subunit with an almost entirely deleted CTD (LS*Δ5) in B-cells. These cells have a defect in global transcription of cellular genes in the presence of α-amanitin. Moreover, pol II harboring LS*Δ5 failed to transcribe up to the promoter-proximal pause sites in thehsp70A and c-fos gene promoters. The results indicate that the CTD is already required for steps that occur before promoter-proximal pausing and maturation of mRNA. The carboxyl-terminal domain (CTD) of the large subunit of mammalian RNA polymerase II contains 52 repeats of a heptapeptide that is the target of a variety of kinases. The hyperphosphorylated CTD recruits important factors for mRNA capping, splicing, and 3′-processing. The role of the CTD for the transcription process in vivo, however, is not yet clear. We have conditionally expressed an α-amanitin-resistant large subunit with an almost entirely deleted CTD (LS*Δ5) in B-cells. These cells have a defect in global transcription of cellular genes in the presence of α-amanitin. Moreover, pol II harboring LS*Δ5 failed to transcribe up to the promoter-proximal pause sites in thehsp70A and c-fos gene promoters. The results indicate that the CTD is already required for steps that occur before promoter-proximal pausing and maturation of mRNA. polymerase II large subunit 12-O-tetradecanoylphorbol-13-acetate kilobase pairs base pair 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole DRB sensitivity-inducing factor hemagglutinin tetracycline cytomegalovirus Eukaryotic mRNA synthesis is catalyzed by the multisubunit RNA polymerase II (pol II).1 The large subunit of pol II (LS) is highly conserved among eukaryotic RNA polymerases and also shows striking homology to the large subunit ofEscherichia coli RNA polymerase (1Uptain S.M. Kane C.M. Chamberlin M.J. Annu. Rev. Biochem. 1997; 66: 117-172Crossref PubMed Scopus (397) Google Scholar). The LS has evolved a particularly structured carboxyl-terminal domain (CTD) that is not present in other RNA polymerases (2Corden J.L. Patturajan M. Trends Biochem. Sci. 1997; 22: 413-416Abstract Full Text PDF PubMed Scopus (148) Google Scholar). This CTD comprises multiple copies of a heptapeptide repeat with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The number of repeats varies from 26/27 in yeast to 52 in mouse and human cells (3Young R.A. Annu. Rev. Biochem. 1991; 60: 689-715Crossref PubMed Scopus (363) Google Scholar). Deletion of more than half of the repeats in yeast and mouse interferes with cell viability (4Bartolomei M.S. Halden N.F. Cullen C.R. Corden J.L. Mol. Cell. Biol. 1988; 8: 330-339Crossref PubMed Scopus (146) Google Scholar, 5Allison L.A. Wong J.K. Fitzpatrick V.D. Moyle M. Ingles C.J. Mol. Cell. Biol. 1988; 8: 321-329Crossref PubMed Scopus (152) Google Scholar, 6Scafe C. Chao D. Lopes J. Hirsch J.P. Henry S. Young R.A. Nature. 1990; 347: 491-494Crossref PubMed Scopus (123) Google Scholar). Mice homozygous for a deletion of 13 repeats are smaller than wild-type littermates and have a high rate of neonatal lethality (7Litingtung Y. Lawler A.M. Sebald S.M. Lee E. Gearhart J.D. Westphal H. Corden J.L. Mol. Gen. Genet. 1999; 261: 100-105Crossref PubMed Scopus (41) Google Scholar), suggesting that CTD is important in regulating growth during mammalian development. In cells, two forms of pol II are detectable containing either a hypophosphorylated (pol IIA) or hyperphosphorylated CTD (pol II0). Although pol IIA is consistently found in the initiation complex, pol II0 is associated with elongating complexes.An increasing number of genes have been shown to be regulated by promoter-proximal pausing of pol II. These genes includeDrosophila hsp70 and hsp26 genes, as well as the mammalian c-myc, c-fos, and immunoglobulin κ genes (8Rougvie A.E. Lis J.T. Cell. 1988; 54: 795-804Abstract Full Text PDF PubMed Scopus (458) Google Scholar, 9Krumm A. Meulia T. Brunvand M. Groudine M. Genes Dev. 1992; 6: 2201-2213Crossref PubMed Scopus (221) Google Scholar, 10Strobl L.J. Eick D. EMBO J. 1992; 11: 3307-3314Crossref PubMed Scopus (144) Google Scholar, 11O'Brien T. Hardin S. Greenleaf A. Lis J.T. Nature. 1994; 370: 75-77Crossref PubMed Scopus (284) Google Scholar, 12Plet A. Eick D. Blanchard J.M. Oncogene. 1995; 10: 319-328PubMed Google Scholar, 13Wolf D.A. Strobl L.J. Pullner A. Eick D. Nucleic Acids Res. 1995; 23: 3373-3379Crossref PubMed Scopus (20) Google Scholar, 14Pinaud S. Mirkovitch J. J. Mol. Biol. 1998; 280: 785-798Crossref PubMed Scopus (38) Google Scholar, 15Raschke E.E. Albert T. Eick D. J. Immunol. 1999; 163: 4375-4382PubMed Google Scholar). The passage of the paused pol II into a processive mode coincides in vivo with hyperphosphorylation of the CTD (11O'Brien T. Hardin S. Greenleaf A. Lis J.T. Nature. 1994; 370: 75-77Crossref PubMed Scopus (284) Google Scholar, 16Weeks J.R. Hardin S.E. Shen J. Lee J.M. Greenleaf A.L. Genes Dev. 1993; 7: 2329-2344Crossref PubMed Scopus (162) Google Scholar).Recent studies suggest that the hyperphosphorylated CTD functions as a platform for the assembly of complexes that cap, splice, cleave, and polyadenylate pre-mRNA (2Corden J.L. Patturajan M. Trends Biochem. Sci. 1997; 22: 413-416Abstract Full Text PDF PubMed Scopus (148) Google Scholar, 17Bentley D. Curr. Opin. Cell Biol. 1999; 11: 347-351Crossref PubMed Scopus (196) Google Scholar, 18Reines D. Conaway R.C. Conaway J.W. Curr. Opin. Cell Biol. 1999; 11: 342-346Crossref PubMed Scopus (69) Google Scholar). Capping of mRNA occurs shortly after transcription initiation (19Coppola J.A. Field A.S. Luse D.S. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 1251-1255Crossref PubMed Scopus (71) Google Scholar), preceding other mRNA processing events such as mRNA splicing and polyadenylation. The capping enzyme is not stably associated with basal transcription factors or the RNA pol II holoenzyme but is directly and specifically recruited to the hyperphosphorylated form of CTD (20Cho E.J. Takagi T. Moore C.R. Buratowski S. Genes Dev. 1997; 11: 3319-3326Crossref PubMed Scopus (369) Google Scholar, 21McCracken S. Fong N. Yankulov K. Ballantyne S. Pan G. Greenblatt J. Patterson S.D. Wickens M. Bentley D.L. Nature. 1997; 385: 357-361Crossref PubMed Scopus (735) Google Scholar, 22McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (428) Google Scholar, 24Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar). Similarly, several components of the splicing machinery (25Kim E. Du L. Bregman D.B. Warren S.L. J. Cell Biol. 1997; 136: 19-28Crossref PubMed Scopus (213) Google Scholar, 26Misteli T. Spector D.L. Mol. Cell. 1999; 3: 697-705Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar) and related factors such as SR proteins and SR-like proteins (27Yuryev 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 (288) Google Scholar, 28Bourquin J.P. Stagljar I. Meier P. Moosmann P. Silke J. Baechi T. Georgiev O. Schaffner W. Nucleic Acids Res. 1997; 25: 2055-2061Crossref PubMed Scopus (88) Google Scholar, 29Patturajan M. Wei X. Berezney R. Corden J.L. Mol. Cell. Biol. 1998; 18: 2406-2415Crossref PubMed Scopus (84) Google Scholar) are recruited to pol II by the hyperphosphorylated CTD. The cleavage-polyadenylation factors CPSF and CstF specifically bind to the hyperphosphorylated CTD and copurify with pol II in a high molecular mass complex (21McCracken S. Fong N. Yankulov K. Ballantyne S. Pan G. Greenblatt J. Patterson S.D. Wickens M. Bentley D.L. Nature. 1997; 385: 357-361Crossref PubMed Scopus (735) Google Scholar), suggesting that polyadenylation factors can be recruited to an RNA 3′-processing signal by pol II, where they dissociate from the polymerase and initiate polyadenylation. In an extension of this model, pol II is required for 3′-processing in vitro in the absence of transcription (30Hirose Y. Manley J.L. Nature. 1998; 395: 93-96Crossref PubMed Scopus (298) Google Scholar, 31Hirose Y. Tacke R. Manley J.L. Genes Dev. 1999; 13: 1234-1239Crossref PubMed Scopus (174) Google Scholar).In addition to pre-mRNA maturation, hyperphosphorylation of CTD appears to play an important role in rendering pol II processive. A positive and a negative elongation factor, implicated in 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) inhibition of transcription elongation, have been identified. DSIF (DRB sensitivity-inducing factor) is a negative elongation factor that renders elongation sensitive to DRB (32Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (557) Google Scholar, 33Wada T. Takagi T. Yamaguchi Y. Watanabe D. Handa H. EMBO J. 1998; 17: 7395-7403Crossref PubMed Scopus (280) Google Scholar, 34Yamaguchi Y. Takagi T. Wada T. Yano K. Furuya A. Sugimoto S. Hasegawa J. Handa H. Cell. 1999; 97: 41-51Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar). It consists of p14 and p160 subunits, which have homology to the yeast Spt4-Spt5 complex (35Hartzog G.A. Wada T. Handa H. Winston F. Genes Dev. 1998; 12: 357-369Crossref PubMed Scopus (370) Google Scholar). p160 shows homology to the bacterial elongation factor NusG (32Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (557) Google Scholar, 33Wada T. Takagi T. Yamaguchi Y. Watanabe D. Handa H. EMBO J. 1998; 17: 7395-7403Crossref PubMed Scopus (280) Google Scholar) and interacts with another negative transcription factor, NELF (36Yamaguchi Y. Wada T. Handa H. Genes Cells. 1998; 3: 9-15Crossref PubMed Scopus (65) Google Scholar). The state of CTD phosphorylation determines the negative action of DSIF on RNA elongation and provides a direct link between DSIF and the positive elongation factor P-TEFb, a CTD-specific kinase (cyclin T/cdk9), which is inhibited by DRB (37Zhu Y. Pe'ery T. Peng J. Ramanathan Y. Marshall N. Marshall T. Amendt B. Mathews M.B. Price D.H. Genes Dev. 1997; 11: 2622-2632Crossref PubMed Scopus (606) Google Scholar, 38Wei P. Garber M.E. Fang S.M. Fischer W.H. Jones K.A. Cell. 1998; 92: 451-462Abstract Full Text Full Text PDF PubMed Scopus (1041) Google Scholar). Other factors,e.g. the Elongator complex, may also bind to pol II in a CTD-dependent manner (39Otero G. Fellows J. Li Y. de Bizemont T. Dirac A.M. Gustafsson C.M. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Mol. Cell. 1999; 3: 109-118Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). Taken together, a huge body of evidence suggests an important role for CTD in activation of RNA elongation and maturation of pre-mRNA in vivo.However, it is not yet clear whether the CTD of pol II is always required for transcriptional initiation in vivo. Several studies showed that pol II with a deleted CTD is transcriptionally active in vitro (Serizawa et al. (40Serizawa H. Conaway J.W. Conaway R.C. Nature. 1993; 363: 371-374Crossref PubMed Scopus (138) Google Scholar)) and initiates and transcribes transiently transfected genes (21McCracken S. Fong N. Yankulov K. Ballantyne S. Pan G. Greenblatt J. Patterson S.D. Wickens M. Bentley D.L. Nature. 1997; 385: 357-361Crossref PubMed Scopus (735) Google Scholar, 41Gerber H.P. Hagmann M. Seipel K. Georgiev O. West M.A. Litingtung Y. Schaffner W. Corden J.L. Nature. 1995; 374: 660-662Crossref PubMed Scopus (136) Google Scholar) as well as the CUP1 gene in yeast (42McNeil J.B. Agah H. Bentley D. Genes Dev. 1998; 12: 2510-2521Crossref PubMed Scopus (85) Google Scholar). Here, we show evidence that pol II with a deleted CTD is defective in global gene transcription and is unable to transcribe up to promoter-proximal pause sites on chromosomal templates.DISCUSSIONWe have established human B-cell lines conditionally expressing the large subunit of pol II to study the effect of CTD deletions on transcription initiation and promoter-proximal pausing. Long term cultures could be established in the presence of α-amanitin from Raji cells expressing LS*wt but not from cells expressing LS*Δ31 and LS*Δ5. Raji cells expressing LS*wt initially run through a crisis but thereafter proliferated quite normally in the presence of α-amanitin, with a doubling time of 30 h. Thus, constitutive expression of LS*wt from a heterologous promoter does not conflict with proliferation and long term survival of Raji cells. Currently we do not know the reason for the initial crisis. A possible reason for the crisis could be the point mutation in LS*wt that confers α-amanitin resistance. Cells expressing this mutant may have an altered gene expression pattern and require an adaptation phase for growth. In contrast, expression of LS*Δ31, even though it initially prolonged survival, and LS*Δ5 failed to replace the endogenous LS in regard to long term survival. This is in line with an earlier report that ratL6 myoblasts expressing LS*Δ31 showed a limited growth in a colony assay, whereas LS*Δ5 failed to support growth (4Bartolomei M.S. Halden N.F. Cullen C.R. Corden J.L. Mol. Cell. Biol. 1988; 8: 330-339Crossref PubMed Scopus (146) Google Scholar).In this study we were particularly interested in the question how deletion of the CTD affects global gene expression in Raji cells. LS*Δ5 has been shown to be defective in enhancer-driven expression of transiently transfected genes (41Gerber H.P. Hagmann M. Seipel K. Georgiev O. West M.A. Litingtung Y. Schaffner W. Corden J.L. Nature. 1995; 374: 660-662Crossref PubMed Scopus (136) Google Scholar). The mutant has also been reported to be unable to facilitate pre-mRNA maturation in transient transfection experiments (21McCracken S. Fong N. Yankulov K. Ballantyne S. Pan G. Greenblatt J. Patterson S.D. Wickens M. Bentley D.L. Nature. 1997; 385: 357-361Crossref PubMed Scopus (735) Google Scholar). However, LS*Δ5 is able to transcribe transiently transfected CMV promoter and SP1-driven promoter constructs (41Gerber H.P. Hagmann M. Seipel K. Georgiev O. West M.A. Litingtung Y. Schaffner W. Corden J.L. Nature. 1995; 374: 660-662Crossref PubMed Scopus (136) Google Scholar). These results could be confirmed for the CMV promoter and in addition for the c-myc promoter in RajiLS*Δ5 cells. pol II with a deleted CTD has also been reported to initiate at theDrosophila hsp70 gene promoter and to transcribe to the promoter-proximal pause site in vitro (53Li B. Weber J.A. Chen Y. Greenleaf A.L. Gilmour D.S. Mol. Cell. Biol. 1996; 16: 5433-5443Crossref PubMed Scopus (39) Google Scholar). Thus, it was not clear whether LS*Δ5 has a general defect in initiation and/or elongation.A first approach to compare steady-state mRNA levels in cells expressing LS*wt and LS*Δ5 turned out to be unsuccessful, because treatment with α-amanitin for 24 h did not lead to significant changes in steady-state mRNA levels. This unexpected result suggested that inhibition of pol II transcription by α-amanitin may lead to a global stabilization of mRNA in Raji cells. Stabilization of mRNA by protein synthesis inhibitors, like cycloheximide, is a well known phenomenon that most likely results from the inhibition of the synthesis of factors required for mRNA degradation. Stabilization of mRNA by transcription inhibitors has been reported for the transferrin receptor gene (54Seiser C. Posch M. Thompson N. Kuhn L.C. J. Biol. Chem. 1995; 270: 29400-29406Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and multidrug resistance genemdr1 (55Kuo M.T. Julian J. Husain F. Song R. Carson D.D. J. Cell. Physiol. 1995; 164: 132-141Crossref PubMed Scopus (15) Google Scholar) but is not yet known to be a general phenomenon. This observation certainly deserves further analysis. Here, this phenomenon made a comparison of mRNAs levels in cells expressing LS*wt and LS*Δ5 impossible.As a consequence we measured the transcription rate of genes in nuclear run-on experiments. More than 500 transcriptionally active genes in phorbol ester-stimulated Raji cells were analyzed. These genes were also found to be transcribed in cells expressing LS*wt but were repressed or strongly reduced in transcription in cells expressing LS*Δ5, indicating that LS*Δ5 has a severe and general defect in transcription in vivo. We did not detect a single gene whose transcription was not severely affected. Notably, the low but significant transcription signals that were still detectable for a few genes in RajiLS*Δ5 but not in RajiLS*mock cells in the presence of α-amanitin indicate that the mutant LS*Δ5 is not transcriptionally dead. This is in agreement with the transient transfection experiments by us and others (21McCracken S. Fong N. Yankulov K. Ballantyne S. Pan G. Greenblatt J. Patterson S.D. Wickens M. Bentley D.L. Nature. 1997; 385: 357-361Crossref PubMed Scopus (735) Google Scholar, 22McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (428) Google Scholar, 41Gerber H.P. Hagmann M. Seipel K. Georgiev O. West M.A. Litingtung Y. Schaffner W. Corden J.L. Nature. 1995; 374: 660-662Crossref PubMed Scopus (136) Google Scholar). The ability of LS*Δ5 to transcribe from some promoters in transfection may rely on the fact that transiently transfected DNA does not establish proper chromatin and may be easily accessible for the transcriptional machinery. Genes packaged in regular chromatin may be less accessible to the transcriptional machinery, particularly if the CTD is deleted. In conclusion, a minimal size of the CTD appears to be required for the transcription of all mammalian genes. However, we cannot rule out that very few of the ∼100,000 estimated genes do not require the function of CTD for its transcription. For example, the CUP1 gene in yeast has been reported to be transcribed quite efficiently by pol II with a deleted CTD (42McNeil J.B. Agah H. Bentley D. Genes Dev. 1998; 12: 2510-2521Crossref PubMed Scopus (85) Google Scholar).What could be the reason that pol II without a CTD is unable to transcribe chromatin-packaged genes? In addition to binding of pre-mRNA maturation factors, CTD may permit recruitment of factors to the transcriptional machinery, either directly or indirectly, that allows transcription of chromatin-packaged DNA templates. Such a factor could be the Elongator complex, for example, which harbors HAT activity and binds to pol II only if the CTD is hyperphosphorylated (39Otero G. Fellows J. Li Y. de Bizemont T. Dirac A.M. Gustafsson C.M. Erdjument-Bromage H. Tempst P. Svejstrup J.Q. Mol. Cell. 1999; 3: 109-118Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar). Alternatively, the phosphorylated CTD may be required to release inhibitory factors from pol II, e.g. DSIF, which interfere with elongation (32Wada T. Takagi T. Yamaguchi Y. Ferdous A. Imai T. Hirose S. Sugimoto S. Yano K. Hartzog G.A. Winston F. Buratowski S. Handa H. Genes Dev. 1998; 12: 343-356Crossref PubMed Scopus (557) Google Scholar, 33Wada T. Takagi T. Yamaguchi Y. Watanabe D. Handa H. EMBO J. 1998; 17: 7395-7403Crossref PubMed Scopus (280) Google Scholar, 36Yamaguchi Y. Wada T. Handa H. Genes Cells. 1998; 3: 9-15Crossref PubMed Scopus (65) Google Scholar). Since phosphorylation of the CTD is assumed to be a critical step in activation of promoter-proximal paused pol II, we asked if the transition from a paused to an elongating pol II is affected if CTD is deleted. At the hsp70A and the c-fos promoters, pol II with a deleted CTD was unable to produce a transcription signal in nuclear run-ons, neither in uninduced nor induced cells. The run-on reactions have been carried out in the presence of Sarkosyl, which has been described to release nucleosomes from DNA (23Scheer U. Cell. 1978; 13: 535-549Abstract Full Text PDF PubMed Scopus (105) Google Scholar) and activate paused pol II (8Rougvie A.E. Lis J.T. Cell. 1988; 54: 795-804Abstract Full Text PDF PubMed Scopus (458) Google Scholar). If pol II with a deleted CTD would be present at the pause site, Sarkosyl should induce its transcriptional activity in nuclear run-on experiments. Therefore, thehsp70A and c-fos genes in Raji cells do not harbor a pol II with a LS*Δ5 at promoter-proximal pause sites. Thus, LS*Δ5 has a defect in initiation or in the very early steps of RNA elongation prior to the polymerase undergoing its conformational change required for elongation. We found evidence that LS*Δ5 may have, dependent on the promoter, a defect at both levels. If the endogenous LS and LS*Δ5 were coexpressed in the absence of α-amanitin, LS*Δ5 showed a dominant-negative effect on transcription of the episomal construct. This suggests that LS*Δ5 can still somehow interact with or bind to the promoter but is unable to elongate. As a consequence the promoter is blocked for binding of endogenous pol II. Interestingly, this dominant-negative effect of LS*Δ5 was not seen for the c-myc, Ig μ, c-fos, and hsp70A genes. For these genes, CTD may already be required for binding of pol II to the promoter. pol II with a deleted CTD cannot compete in binding. From these data we conclude that the CTD not only fulfills important tasks in RNA elongation and maturation of pre-mRNA but also in initiation and/or early elongation. Eukaryotic mRNA synthesis is catalyzed by the multisubunit RNA polymerase II (pol II).1 The large subunit of pol II (LS) is highly conserved among eukaryotic RNA polymerases and also shows striking homology to the large subunit ofEscherichia coli RNA polymerase (1Uptain S.M. Kane C.M. Chamberlin M.J. Annu. Rev. Biochem. 1997; 66: 117-172Crossref PubMed Scopus (397) Google Scholar). The LS has evolved a particularly structured carboxyl-terminal domain (CTD) that is not present in other RNA polymerases (2Corden J.L. Patturajan M. Trends Biochem. Sci. 1997; 22: 413-416Abstract Full Text PDF PubMed Scopus (148) Google Scholar). This CTD comprises multiple copies of a heptapeptide repeat with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The number of repeats varies from 26/27 in yeast to 52 in mouse and human cells (3Young R.A. Annu. Rev. Biochem. 1991; 60: 689-715Crossref PubMed Scopus (363) Google Scholar). Deletion of more than half of the repeats in yeast and mouse interferes with cell viability (4Bartolomei M.S. Halden N.F. Cullen C.R. Corden J.L. Mol. Cell. Biol. 1988; 8: 330-339Crossref PubMed Scopus (146) Google Scholar, 5Allison L.A. Wong J.K. Fitzpatrick V.D. Moyle M. Ingles C.J. Mol. Cell. Biol. 1988; 8: 321-329Crossref PubMed Scopus (152) Google Scholar, 6Scafe C. Chao D. Lopes J. Hirsch J.P. Henry S. Young R.A. Nature. 1990; 347: 491-494Crossref PubMed Scopus (123) Google Scholar). Mice homozygous for a deletion of 13 repeats are smaller than wild-type littermates and have a high rate of neonatal lethality (7Litingtung Y. Lawler A.M. Sebald S.M. Lee E. Gearhart J.D. Westphal H. Corden J.L. Mol. Gen. Genet. 1999; 261: 100-105Crossref PubMed Scopus (41) Google Scholar), suggesting that CTD is important in regulating growth during mammalian development. In cells, two forms of pol II are detectable containing either a hypophosphorylated (pol IIA) or hyperphosphorylated CTD (pol II0). Although pol IIA is consistently found in the initiation complex, pol II0 is associated with elongating complexes. An increasing number of genes have been shown to be regulated by promoter-proximal pausing of pol II. These genes includeDrosophila hsp70 and hsp26 genes, as well as the mammalian c-myc, c-fos, and immunoglobulin κ genes (8Rougvie A.E. Lis J.T. Cell. 1988; 54: 795-804Abstract Full Text PDF PubMed Scopus (458) Google Scholar, 9Krumm A. Meulia T. Brunvand M. Groudine M. Genes Dev. 1992; 6: 2201-2213Crossref PubMed Scopus (221) Google Scholar, 10Strobl L.J. Eick D. EMBO J. 1992; 11: 3307-3314Crossref PubMed Scopus (144) Google Scholar, 11O'Brien T. Hardin S. Greenleaf A. Lis J.T. Nature. 1994; 370: 75-77Crossref PubMed Scopus (284) Google Scholar, 12Plet A. Eick D. Blanchard J.M. Oncogene. 1995; 10: 319-328PubMed Google Scholar, 13Wolf D.A. Strobl L.J. Pullner A. Eick D. Nucleic Acids Res. 1995; 23: 3373-3379Crossref PubMed Scopus (20) Google Scholar, 14Pinaud S. Mirkovitch J. J. Mol. Biol. 1998; 280: 785-798Crossref PubMed Scopus (38) Google Scholar, 15Raschke E.E. Albert T. Eick D. J. Immunol. 1999; 163: 4375-4382PubMed Google Scholar). The passage of the paused pol II into a processive mode coincides in vivo with hyperphosphorylation of the CTD (11O'Brien T. Hardin S. Greenleaf A. Lis J.T. Nature. 1994; 370: 75-77Crossref PubMed Scopus (284) Google Scholar, 16Weeks J.R. Hardin S.E. Shen J. Lee J.M. Greenleaf A.L. Genes Dev. 1993; 7: 2329-2344Crossref PubMed Scopus (162) Google Scholar). Recent studies suggest that the hyperphosphorylated CTD functions as a platform for the assembly of complexes that cap, splice, cleave, and polyadenylate pre-mRNA (2Corden J.L. Patturajan M. Trends Biochem. Sci. 1997; 22: 413-416Abstract Full Text PDF PubMed Scopus (148) Google Scholar, 17Bentley D. Curr. Opin. Cell Biol. 1999; 11: 347-351Crossref PubMed Scopus (196) Google Scholar, 18Reines D. Conaway R.C. Conaway J.W. Curr. Opin. Cell Biol. 1999; 11: 342-346Crossref PubMed Scopus (69) Google Scholar). Capping of mRNA occurs shortly after transcription initiation (19Coppola J.A. Field A.S. Luse D.S. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 1251-1255Crossref PubMed Scopus (71) Google Scholar), preceding other mRNA processing events such as mRNA splicing and polyadenylation. The capping enzyme is not stably associated with basal transcription factors or the RNA pol II holoenzyme but is directly and specifically recruited to the hyperphosphorylated form of CTD (20Cho E.J. Takagi T. Moore C.R. Buratowski S. Genes Dev. 1997; 11: 3319-3326Crossref PubMed Scopus (369) Google Scholar, 21McCracken S. Fong N. Yankulov K. Ballantyne S. Pan G. Greenblatt J. Patterson S.D. Wickens M. Bentley D.L. Nature. 1997; 385: 357-361Crossref PubMed Scopus (735) Google Scholar, 22McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (428) Google Scholar, 24Ho C.K. Schwer B. Shuman S. Mol. Cell. Biol. 1998; 18: 5189-5198Crossref PubMed Google Scholar). Similarly, several components of the splicing machinery (25Kim E. Du L. Bregman D.B. Warren S.L. J. Cell Biol. 1997; 136: 19-28Crossref PubMed Scopus (213) Google Scholar, 26Misteli T. Spector D.L. Mol. Cell. 1999; 3: 697-705Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar) and related factors such as SR proteins and SR-like proteins (27Yuryev 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 (288) Google Scholar, 28Bourquin J.P. Stagljar I. Meier P. Moosmann P. Silke J. Baechi T. Georgiev O. Schaffner W. Nucleic Acids Res. 1997; 25: 2055-2061Crossref PubMed Scopus (88) Google Scholar, 29Patturajan M. Wei X. 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