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• W1995771026 abstract "Cycloheximide inhibits ribosomal DNA (rDNA) transcription in vivo. The mouse homologue of yeast Rrn3, a polymerase-associated transcription initiation factor, can complement extracts from cycloheximide-treated mammalian cells. Cycloheximide inhibits the phosphorylation of Rrn3 and causes its dissociation from RNA polymerase I. Rrn3 interacts with the rpa43 subunit of RNA polymerase I, and treatment with cycloheximide inhibits the formation of a Rrn3·rpa43 complex in vivo. Rrn3 produced in Sf9 cells but not in bacteria interacts with rpa43 in vitro, and such interaction is dependent upon the phosphorylation state of Rrn3. Significantly, neither dephosphorylated Rrn3 nor Rrn3 produced in Escherichia coli can restore transcription by extracts from cycloheximide-treated cells. These results suggest that the phosphorylation state of Rrn3 regulates rDNA transcription by determining the steady-state concentration of the Rrn3·RNA polymerase I complex within the nucleolus. Cycloheximide inhibits ribosomal DNA (rDNA) transcription in vivo. The mouse homologue of yeast Rrn3, a polymerase-associated transcription initiation factor, can complement extracts from cycloheximide-treated mammalian cells. Cycloheximide inhibits the phosphorylation of Rrn3 and causes its dissociation from RNA polymerase I. Rrn3 interacts with the rpa43 subunit of RNA polymerase I, and treatment with cycloheximide inhibits the formation of a Rrn3·rpa43 complex in vivo. Rrn3 produced in Sf9 cells but not in bacteria interacts with rpa43 in vitro, and such interaction is dependent upon the phosphorylation state of Rrn3. Significantly, neither dephosphorylated Rrn3 nor Rrn3 produced in Escherichia coli can restore transcription by extracts from cycloheximide-treated cells. These results suggest that the phosphorylation state of Rrn3 regulates rDNA transcription by determining the steady-state concentration of the Rrn3·RNA polymerase I complex within the nucleolus. RNA polymerase I ribosomal RNA human Rrn3 dexamethasone cycloheximide selectivity factor I 68-kDa subunit of SL1 upstream binding factor transcription factor 1C transcription initiation factor 1A mouse homologue of yrpa43 β′ subunit of RNA polymerase I β subunit of RNA polymerase I fluorescencein situ hybridization external transcribed spacer phosphate-buffered saline bovine serum albumin nucleotide(s) nickel-nitrilotriacetic acid bacterial alkaline phosphatase calf intestinal alkaline phosphatase green fluorescent protein In the early 1970s Feigelson and colleagues (1Yu F.L. Feigelson P. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 2177-2180Crossref PubMed Scopus (97) Google Scholar, 2Yu F.L. Feigelson P. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 2833-2837Crossref PubMed Scopus (104) Google Scholar, 3Lampert A. Feigelson P. Biochem. Biophys. Res. Commun. 1974; 58: 1030-1038Crossref PubMed Scopus (53) Google Scholar) reported that cycloheximide caused a rapid cessation of nucleolar RNA synthesis (ribosomal DNA transcription) and concluded that a rapidly turning over protein was required for RNA polymerase I (pol I)1 activity in vivo. Subsequent studies have demonstrated that transcription by RNA polymerase I is subject to regulation at many levels (4Hannan K.M. Hannan R.D. Rothblum L.I. Front. Biosci. 1998; 3: d376-d398Crossref PubMed Google Scholar, 5Grummt I. Prog. Nucleic Acids Res. Mol. Biol. 1999; 62: 109-154Crossref PubMed Scopus (204) Google Scholar). At least three, and possibly more, polymerase-associated proteins, TIF-IA, Factor C*, and TFIC (6Cavanaugh A.H. Gokal P.K. Lawther R.P. Thompson E.A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 718-721Crossref PubMed Scopus (33) Google Scholar, 7Buttgereit D. Pflugfelder G. Grummt I. Nucleic Acids Res. 1985; 13: 8165-8180Crossref PubMed Scopus (76) Google Scholar, 8Tower J. Sollner-Webb B. Cell. 1987; 50: 873-883Abstract Full Text PDF PubMed Scopus (70) Google Scholar), have been demonstrated to contribute to the regulation of rDNA transcription. TIF-IA and Factor C* were identified as factors that were required for the complementation of extracts of quiescent or cycloheximide-treated cells. TFIC was identified as that activity required to reconstitute transcription by extracts of glucocorticoid-treated P1798 cells. This lymphosarcoma cell line exits the cell cycle in response to the synthetic glucocorticoid dexamethasone (DEX) (6Cavanaugh A.H. Gokal P.K. Lawther R.P. Thompson E.A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 718-721Crossref PubMed Scopus (33) Google Scholar). Interestingly, TIF-IA, Factor C*, and TFIC shared several properties, including a tight association with the core polymerase (8Tower J. Sollner-Webb B. Cell. 1987; 50: 873-883Abstract Full Text PDF PubMed Scopus (70) Google Scholar, 9Mahajan P.B. Thompson E.A. J. Biol. Chem. 1990; 265: 16225-16233Abstract Full Text PDF PubMed Google Scholar, 10Schnapp A. Pfleiderer C. Rosenbauer H. Grummt I. EMBO J. 1990; 9: 2857-2863Crossref PubMed Scopus (93) Google Scholar). TIF-IA and TFIC were purified and consisted of different polypeptides (10Schnapp A. Pfleiderer C. Rosenbauer H. Grummt I. EMBO J. 1990; 9: 2857-2863Crossref PubMed Scopus (93) Google Scholar, 11Schnapp A. Schnapp G. Erny B. Grummt I. Mol. Cell. Biol. 1993; 13: 6723-6732Crossref PubMed Scopus (69) Google Scholar). However, the lack of immunological and molecular tools precluded a definitive statement that TIF-IA and TFIC were the same or different proteins (reviewed in Refs. 4Hannan K.M. Hannan R.D. Rothblum L.I. Front. Biosci. 1998; 3: d376-d398Crossref PubMed Google Scholar and 5Grummt I. Prog. Nucleic Acids Res. Mol. Biol. 1999; 62: 109-154Crossref PubMed Scopus (204) Google Scholar).The formation of the stable preinitiation complex in yeast requires an interaction between the upstream activating factor bound to the upstream promoter element and core factor, bound to the core promoter element. This complex then recruits transcriptionally competent RNA polymerase I to the transcription initiation site (Ref. 12Aprikian P. Moorefield B. Reeder R.H. Mol. Cell. Biol. 2001; 21: 4847-4855Crossref PubMed Scopus (55) Google Scholar and references therein). Mechanistically, Rrn3 appears to “bridge” the polymerase and transcription initiation complexes (13Thuriaux P. Mariotte S. Buhler J.M. Sentenac A., Vu, L. Lee B.S. Nomura M. J. Biol. Chem. 1995; 270: 24252-24257Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 14Yamamoto R. Nogi Y. Dodd J. Nomura M. EMBO J. 1996; 15: 3964-3973Crossref PubMed Scopus (102) Google Scholar, 15Peyroche G. Milkereit P. Bischler N. Tschochner H. Schultz P. Sentenac A. Carles C. Riva M. EMBO J. 2000; 19: 5473-5482Crossref PubMed Scopus (138) Google Scholar). Thus, only pol I molecules in complex with Rrn3 are able to recognize the preinitiation complex and initiate transcription.Studies comparing the state of RNA polymerase I in growing and stationary yeast cells demonstrated that ∼2% of the pol I in whole cell extracts was capable of initiating transcription in vitro (16Milkereit P. Tschochner H. EMBO J. 1998; 17: 3692-3703Crossref PubMed Scopus (116) Google Scholar). This correlated with the observation that the association of Rrn3 with pol I corresponded to the growth state of the cells and was confirmed by the observation that transcriptionally active pol I was associated with Rrn3 (16Milkereit P. Tschochner H. EMBO J. 1998; 17: 3692-3703Crossref PubMed Scopus (116) Google Scholar, 17Keener J. Josaitis C. Dodd J. Nomura M. J. Biol. Chem. 1998; 272: 33795-33802Abstract Full Text Full Text PDF Scopus (95) Google Scholar). Milkereit and Tschochner (16Milkereit P. Tschochner H. EMBO J. 1998; 17: 3692-3703Crossref PubMed Scopus (116) Google Scholar) demonstrated that the association of Rrn3 with pol I was independent of either the total pol I or total Rrn3 content but varied with the growth rate of the cells. This could reflect alterations in the state of Rrn3 and/or pol I that would shift the equilibrium between free Rrn3 and Rrn3 associated with pol I. Fathet al. (18Fath S. Milkereit P. Peyroche G. Riva M. Carles C. Tschochner H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14334-14339Crossref PubMed Scopus (61) Google Scholar) have recently reported that this equilibrium is modulated by growth-related phosphorylation of specific sites of yeast RNA polymerase I.Moorefield et al. (19Moorefield B. Greene E.A. Reeder R.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4724-4729Crossref PubMed Scopus (85) Google Scholar) demonstrated that hRrn3 could complement yeast Rrn3 mutants, and there is strong evidence that mammalian Rrn3 is the equivalent of TIF-IA (20Bodem J. Dobreva G. Hoffmann-Rohrer U. Iben S. Zentgraf H. Delius H. Vingron M. Grummt I. EMBO Rep. 2000; 1: 171-175Crossref PubMed Scopus (112) Google Scholar). Recombinant mammalian Rrn3 has been shown to complement extracts from cycloheximide-treated cells (20Bodem J. Dobreva G. Hoffmann-Rohrer U. Iben S. Zentgraf H. Delius H. Vingron M. Grummt I. EMBO Rep. 2000; 1: 171-175Crossref PubMed Scopus (112) Google Scholar), suggesting that Rrn3 was the rapidly turning over activity originally identified by Feigelson's laboratory (3Lampert A. Feigelson P. Biochem. Biophys. Res. Commun. 1974; 58: 1030-1038Crossref PubMed Scopus (53) Google Scholar). In addition, mammalian Rrn3 has been shown to interact with SL1 and would then serve as the bridge between RNA polymerase I and the preinitiation complex on the rDNA promoter (21Miller G. Panov K.I. Friedrich J.K. Trinkle-Mulcahy L. Lamond A.I. Zomerdijk J.C. EMBO J. 2001; 20: 1373-1382Crossref PubMed Scopus (146) Google Scholar). However, the mechanism by which Rrn3 activity is inhibited by CHX has not been identified. Moreover, earlier studies on TIF-IA suggested that it did not dissociate from RNA polymerase I (10Schnapp A. Pfleiderer C. Rosenbauer H. Grummt I. EMBO J. 1990; 9: 2857-2863Crossref PubMed Scopus (93) Google Scholar). Thus, the role of Rrn3 in mammalian rDNA transcription and its mode of regulation needed to be examined.We report here that the mammalian homologue of Rrn3 plays a crucial role in rDNA transcription. We have found that Rrn3 can complement extracts of cells treated with CHX but not extracts of DEX-treated P1798 cells, demonstrating that TIF-IA/Rrn3 and TFIC are not the same activities. We have confirmed that hRrn3 can interact with both the core subunits of RNA polymerase I and SL1. Although the Rrn3-SL1 interaction is not affected by CHX, the interaction between Rrn3 and pol I is inhibited. Moreover, we demonstrate that the Rrn3·pol I interaction is, at least in part, mediated by the mouse homologue of rpa43 as it is in Saccharomyces cerevisiae (15Peyroche G. Milkereit P. Bischler N. Tschochner H. Schultz P. Sentenac A. Carles C. Riva M. EMBO J. 2000; 19: 5473-5482Crossref PubMed Scopus (138) Google Scholar). However, the Rrn3·pol I interaction is regulated differently in mammals than in yeast. Treatment with cycloheximide inhibits Rrn3 phosphorylation and is associated with the dissociation of Rrn3 from its complex with RNA polymerase I and the inhibition of the formation of an Rrn3·rpa43 complex in vivo. The interaction between Rrn3 and rpa43 was confirmed using purified recombinant proteins in vitro. Using this in vitro model, we found that dephosphorylation of Rrn3 weakened the interaction between Rrn3 and rpa43. The importance of the role of Rrn3 phosphorylation was confirmed by the observation that dephosphorylated Rrn3 could not reconstitute transcription when added to extracts from cycloheximide-treated cells.DISCUSSIONWe have established that phosphorylation of mammalian Rrn3 regulates its role in rDNA transcription. We have confirmed that the ability of TIF-IA/Rrn3 to function in transcription is inhibited when mammalian cells are treated with CHX. In addition, we demonstrated that Rrn3/TIF-IA is insufficient to reconstitute rDNA transcription when added to extracts from dexamethasone-treated P1798 cells. Therefore, we conclude that Rrn3/TIF-IA and TFIC are not the same factors. We provide several lines of evidence that the phosphorylation status of mammalian Rrn3 is critical for its function. We have shown that treatment of cells with CHX leads to a rapid decrease in the phosphorylation status of Rrn3 and to the dissociation of Rrn3 from core RNA polymerase I. In contrast, CHX did not significantly inhibit the ability of Rrn3 to interact with TAFI68. Our investigation of the interaction of Rrn3 with pol I demonstrated that mammalian Rrn3 interacts with mammalian rpa43 and that this interaction is inhibited when cells are treated with CHX. Moreover, we demonstrated that the interaction of Rrn3 with rpa43 in vitro is inhibited when Rrn3 is dephosphorylated and that treatment with phosphatase inhibits the ability of Rrn3 to reconstitute transcription by extracts of CHX-treated cells. Furthermore, our observation (data not shown) that the distribution of Rrn3 becomes dispersed upon cycloheximide treatment is reminiscent of the morphological alternations of the nucleolus observed after treatment with the kinase inhibitor (5,6-dichloro-β-d-ribofuranosyl-benzimidazole) DRB (30Chen D. Huang S. J. Cell Biol. 2001; 153: 169-176Crossref PubMed Scopus (267) Google Scholar, 31Haaf T. Ward DC. Exp. Cell Res. 1996; 224: 163-173Crossref PubMed Scopus (133) Google Scholar, 32Panse S.L. Masson C. Heliot L. Chassery J.M. Hernandez-Verdun D. J. Cell Sci. 1999; 112: 2145-2154PubMed Google Scholar). This finding is consistent with our model that inhibition of phosphorylation of Rrn3 inhibits its interaction with pol I via rpa43.The model presented in Fig. 7 summarizes the findings of the present report and incorporates several other findings. First, the model demonstrates a requirement for Rrn3 phosphorylation to interact with RNA polymerase I and to allow pol I to productively recognize the preinitiation complex formed by SL1 and UBF on the rDNA promoter. This does not preclude the possibility that RNA polymerase I might interact with the preinitiation complex in the absence of Rrn3. It is formally possible that TFIC serves a parallel function to that served by Rrn3 and might be capable of facilitating an “incomplete” complex between RNA polymerase I and the preinitiation complex formed by SL1 and UBF. Second, the model incorporates observations made by Brun et al. (58Brun R.P. Ryan K. Sollner-Webb B. Mol. Cell. Biol. 1994; 14: 5010-5021Crossref PubMed Scopus (35) Google Scholar) as well as some made by Aprikian et al. (12Aprikian P. Moorefield B. Reeder R.H. Mol. Cell. Biol. 2001; 21: 4847-4855Crossref PubMed Scopus (55) Google Scholar). These authors have demonstrated that Factor C* or the Rrn3·pol I complex dissociates during transcription, and Rrn3 loses its capacity to participate in subsequent rounds of initiation. Third, the model suggests that dephosphorylated Rrn3 would remain complexed with SL1. Although this would be consistent with our fluorescence recovery after photobleaching data, because it would reduce the rate of Rrn3 diffusion, it would appear to contradict the model proposed by Aprikian et al. (12Aprikian P. Moorefield B. Reeder R.H. Mol. Cell. Biol. 2001; 21: 4847-4855Crossref PubMed Scopus (55) Google Scholar).The evidence accumulated to date indicates that the activity of nearly every component of the mammalian rDNA transcriptional apparatus is subject to regulation and that in most cases there are multiple mechanisms for regulating their activity. UBF activity can be regulated by phosphorylation (33O'Mahony D.J. Smith S.D. Xie W. Rothblum L.I. Nucleic Acids Res. 1992; 20: 1301-1308Crossref PubMed Scopus (61) Google Scholar, 34O'Mahony D.J. Xie W.-Q. Smith S.D. Singer H.A. Rothblum L.I. J. Biol. Chem. 1992; 267: 35-38Abstract Full Text PDF PubMed Google Scholar, 35Voit R. Kuhn A. Sander E.E. Grummt I. Nucleic Acids Res. 1995; 23: 2593-2599Crossref PubMed Scopus (97) Google Scholar, 36Klein J. Grummt I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6096-6101Crossref PubMed Scopus (139) Google Scholar, 37Zhai W. Comai L. Mol. Cell. Biol. 2000; 20: 5930-5938Crossref PubMed Scopus (224) Google Scholar, 38Stefanovsky V.Y. Pelletier G. Hannan R.D. Gagnon-Kugler T. Rothblum L.I. Moss T. Mol. Cell. 2001; 8: 1063-1073Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), acetylation (39Pelletier G. Stefanovsky V.Y. Faubladier M. Hirschler-Laszkiewicz I. Savard J. Rothblum L.I. Côté J. Moss T. Mol. Cell. 2000; 6: 1059-1066Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 40Hirschler-Laszkiewicz I. Cavanaugh A., Hu, Q. Catania J. Avantaggiati M.L. Rothblum L.I. Nucleic Acids Res. 2001; 29: 4114-4124Crossref PubMed Scopus (60) Google Scholar), and sequestration in complex with proteins such as Rb and p130 (27Hannan K.M. Hannan R. Smith S. Jefferson L. Lun M. Rothblum L.I. Oncogene. 2000; 19: 4988-4999Crossref PubMed Scopus (107) Google Scholar, 37Zhai W. Comai L. Mol. Cell. Biol. 2000; 20: 5930-5938Crossref PubMed Scopus (224) Google Scholar, 41Cavanaugh A.H. Hempel W.M. Taylor L.J. Rogalsky V. Todorov G. Rothblum L.I. Nature. 1995; 374: 177-180Crossref PubMed Scopus (290) Google Scholar, 42Voit R. Schafer K. Grummt I. Mol. Cell. Biol. 1997; 17: 4230-4237Crossref PubMed Scopus (137) Google Scholar, 43Ciarmatori S. Scott P.H. Sutcliffe J.E. McLees A. Alzuherri H.M. Dannenberg J.H. te Riele H. Grummt I. Voit R. White R.J. Mol. Cell. Biol. 2001; 21: 5806-5814Crossref PubMed Scopus (67) Google Scholar, 44Hannan K.M. Kennedy B.K. Cavanaugh A.H. Hannan R.D. Hirschler-Laszkiewicz I. Jefferson L. Rothblum L.I. Oncogene. 2000; 19: 3487-3497Crossref PubMed Scopus (71) Google Scholar). The amount of UBF present in the cell is also regulated by altering the level of expression of the UBF gene (29Hannan R.D. Luyken J. Rothblum L.I. J. Biol. Chem. 1996; 271: 3213-3220Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Similarly, SL1 is subject to regulation (45Comai L. Song Y. Tan C. Bui T. Cell Growth Diff. 2000; 11: 63-70PubMed Google Scholar) via post-translational modifications, including phosphorylation and acetylation (46Muth V. Nadaud S. Grummt I. Voit R. EMBO J. 2001; 20: 1353-1362Crossref PubMed Scopus (175) Google Scholar). Moreover, there is evidence for cell cycle-specific patterns of phosphorylation of SL1 (47Heix J. Vente A. Voit R. Budde A. Michaelidis T. Grummt I. EMBO J. 1998; 17: 7373-7381Crossref PubMed Scopus (132) Google Scholar, 48Kuhn A. Vente A. Doree M. Grummt I. J. Mol. Biol. 1998; 284: 1-5Crossref PubMed Scopus (46) Google Scholar), UBF (36Klein J. Grummt I. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6096-6101Crossref PubMed Scopus (139) Google Scholar, 49Voit R. Hoffmann M. Grummt I. EMBO J. 1999; 18: 1891-1899Crossref PubMed Scopus (157) Google Scholar), and TTF1 (50Sirri V. Roussel P. Hernandez-Verdun D. J. Cell Sci. 1999; 112: 3259-3268PubMed Google Scholar, 51Sirri V. Roussel P. Hernandez-Verdun D. J. Cell Biol. 2000; 148: 259-270Crossref PubMed Scopus (81) Google Scholar) that correlate with mitotic silencing of rDNA transcription. Similarly, studies on RNA polymerase I demonstrate that pol I activity may be subject to regulation through multiple mechanisms affecting both its catalytic activity and ability to initiate transcription (reviewed in Refs. 5Grummt I. Prog. Nucleic Acids Res. Mol. Biol. 1999; 62: 109-154Crossref PubMed Scopus (204) Google Scholar, 18Fath S. Milkereit P. Peyroche G. Riva M. Carles C. Tschochner H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14334-14339Crossref PubMed Scopus (61) Google Scholar, and 22Hannan R.D. Hempel W.M. Cavanaugh A. Arino T. Dimitrov S.I. Moss T. Rothblum L. J. Biol. Chem. 1998; 273: 1257-1267Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar).Studies on the inactivation of rDNA transcription during encystment of Acanthamoeba castellanii demonstrated that inactivation was the result of a modification of RNA polymerase I (52Detke S. Paule M.R. Arch. Biochem. Biophys. 1978; 185: 333-343Crossref PubMed Scopus (10) Google Scholar, 53Matthews J.L. Zwick M.G. Paule M.R. Mol. Cell. Biol. 1995; 15: 3327-3335Crossref PubMed Scopus (18) Google Scholar). Similarly, studies on the regulation of rDNA transcription in mammalian cells undergoing serum starvation or P1798 cells exposed to DEX demonstrated that a polymerase-associated factor was responsible for the inhibition of rDNA transcription. Interestingly, two laboratories were able to separate what appeared to be the same activity from RNA polymerase I (9Mahajan P.B. Thompson E.A. J. Biol. Chem. 1990; 265: 16225-16233Abstract Full Text PDF PubMed Google Scholar, 10Schnapp A. Pfleiderer C. Rosenbauer H. Grummt I. EMBO J. 1990; 9: 2857-2863Crossref PubMed Scopus (93) Google Scholar). Our results demonstrate that the heat-treated extracts used by Thompson's laboratory (9Mahajan P.B. Thompson E.A. J. Biol. Chem. 1990; 265: 16225-16233Abstract Full Text PDF PubMed Google Scholar) contain both Rrn3/TIF-IA and TFIC and that Rrn3/TIF-IA and TFIC are not the same factors.The model for rDNA transcription in S. cerevisiae proposes that the binding of the multisubunit complex (upstream activation factor) to the upstream promoter element is required to recruit the core factor to the promoter (12Aprikian P. Moorefield B. Reeder R.H. Mol. Cell. Biol. 2001; 21: 4847-4855Crossref PubMed Scopus (55) Google Scholar, 17Keener J. Josaitis C. Dodd J. Nomura M. J. Biol. Chem. 1998; 272: 33795-33802Abstract Full Text Full Text PDF Scopus (95) Google Scholar, 54Siddiqi I. Keener J., Vu, L. Nomura M. Mol. Cell. Biol. 2001; 21: 2292-2297Crossref PubMed Scopus (16) Google Scholar). In turn, the core factor recruits the transcriptionally competent form of pol I to the transcription initiation site. In this model for transcription initiation, Rrn3 functions as the bridge between pol I and the core factor. In yeast, Rrn3 is associated with a small fraction of the RNA polymerase I in the cell, and it is that fraction that is competent for specific initiation of rDNA transcription. This is similar to the report of Tower and Sollner-Webb (8Tower J. Sollner-Webb B. Cell. 1987; 50: 873-883Abstract Full Text PDF PubMed Scopus (70) Google Scholar) who demonstrated the existence of two biochemically definable forms of pol I in mammalian cells; one that was capable of initiating transcription and one that could not. In stationary yeast cells, in which rDNA transcription has been down-regulated, there is a decrease in the association of Rrn3 and pol I, although there is no decrease in the amount of free Rrn3 or pol I (16Milkereit P. Tschochner H. EMBO J. 1998; 17: 3692-3703Crossref PubMed Scopus (116) Google Scholar). Indeed, Milkereit and Tschochner (16Milkereit P. Tschochner H. EMBO J. 1998; 17: 3692-3703Crossref PubMed Scopus (116) Google Scholar) demonstrated that the addition of purified pol I·Rrn3 complex from growing cells to an inactive fraction (PA600s) from stationary cells restored transcription. Interestingly, neither initiation-inactive pol I nor recombinant Rrn3 could complement extracts from stationary cells.Fath et al. (18Fath S. Milkereit P. Peyroche G. Riva M. Carles C. Tschochner H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14334-14339Crossref PubMed Scopus (61) Google Scholar) examined the model that different post-translational modifications have to exist in either Rrn3 and/or RNA polymerase I to regulate the formation of transcriptionally competent RNA polymerase I. They demonstrated that phosphorylation is required to maintain a stable and transcriptionally active pol I·Rrn3 complex. They reported that, although yeast Rrn3 is a phosphoprotein, nonphosphorylated Rrn3 and Rrn3 produced in bacteria were able to participate in the formation of the transcriptionally competent RNA polymerase I·Rrn3 complex. Moreover, they demonstrated that treatment of yeast RNA polymerase I with alkaline phosphatase abrogated its ability to interact with Rrn3 and inhibited the formation of transcriptionally competent pol I. These observations led to the model that phosphorylation/dephosphorylation at specific pol I sites mediates the interaction with Rrn3 and the ability of pol I to initiate rDNA transcription. Although structural and genetic studies indicate that yeast Rrn3 interacts with the 43-kDa subunit of pol I (rpa43), it is not yet known if the phosphorylation status of rpa43 regulates the interaction of Rrn3 with rpa43.In contrast, our results led to the conclusion that phosphorylation of Rrn3 is required for the formation of the Rrn3·pol I complex that is capable of transcription initiation. Although this model for regulating rDNA transcription would be different from that proposed by Fathet al. (18Fath S. Milkereit P. Peyroche G. Riva M. Carles C. Tschochner H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14334-14339Crossref PubMed Scopus (61) Google Scholar), it is consistent with the general model that the formation of the Rrn3·pol I complex is essential for rDNA transcription. Moreover, most of the studies on the regulation of polymerase-associated factors required for rDNA transcription in mammalian cells are consistent with a model wherein Rrn3, and not pol I, would be the target of post-translational modification, as in the experiments presented herein and those of Bodem et al.(20Bodem J. Dobreva G. Hoffmann-Rohrer U. Iben S. Zentgraf H. Delius H. Vingron M. Grummt I. EMBO Rep. 2000; 1: 171-175Crossref PubMed Scopus (112) Google Scholar). We have demonstrated that the addition of recombinant Rrn3 from Sf9 cells to extracts of CHX-treated cells is sufficient to reconstitute transcription. If the post-translational modification of pol I was responsible for the inactivation of transcription in that system, then the addition of Rrn3 would not be sufficient to reconstitute transcription.Our observation, that serum starvation significantly reduces the phosphorylation state of RNA polymerase I in cultured rat hepatoma cells (22Hannan R.D. Hempel W.M. Cavanaugh A. Arino T. Dimitrov S.I. Moss T. Rothblum L. J. Biol. Chem. 1998; 273: 1257-1267Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), is consistent with the formal possibility that the post-translational modification of pol I might also contribute to the interaction of mammalian Rrn3 with pol I. However, only the A194 subunit of mammalian pol I has been definitively demonstrated to be phosphorylated (Ref. 22Hannan R.D. Hempel W.M. Cavanaugh A. Arino T. Dimitrov S.I. Moss T. Rothblum L. J. Biol. Chem. 1998; 273: 1257-1267Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar and discussion therein), and the results from studies on yeast and mammalian RNA polymerase I indicate that Rrn3 interacts with rpa43 and not A194. It is possible that additional subunits contribute to the stability of the Rrn3·pol I complex, and it is interesting to note that several of the yeast RNA polymerase I subunits are phosphorylated, including A190, A34.5, A23, A19, and A43 (55Breant B. Buhler J.-M. Sentenac A. Fromageot P. Eur. J. Biochem. 1983; 130: 247-251Crossref PubMed Scopus (34) Google Scholar, 56Bell G.I. Valenzuela P. Rutter W.J. J. Biol. Chem. 1977; 252: 3082-3091Abstract Full Text PDF PubMed Google Scholar, 57Buhler J.-M. Iborra F. Sentenac A. Fromageot P. FEBS Lett. 1976; 72: 37-41Crossref PubMed Scopus (32) Google Scholar). However, in the absence of evidence to suggest that Rrn3 interacts with additional subunits, our studies have focused on the interaction between Rrn3 and rpa43. The results obtained in those experiments are consistent with the model that the phosphorylation state of Rrn3 determines the formation of the Rrn3·pol I complex in mammalian cells. The determination of the modified residues in Rrn3 and the correlation of the state of modification with the ability of Rrn3 to interact with pol I will provide an important tool to understanding the mechanism of rDNA transcription. In the early 1970s Feigelson and colleagues (1Yu F.L. Feigelson P. Proc. Natl. Acad. Sci. U. S. A. 1971; 68: 2177-2180Crossref PubMed Scopus (97) Google Scholar, 2Yu F.L. Feigelson P. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 2833-2837Crossref PubMed Scopus (104) Google Scholar, 3Lampert A. Feigelson P. Biochem. Biophys. Res. Commun. 1974; 58: 1030-1038Crossref PubMed Scopus (53) Google Scholar) reported that cycloheximide caused a rapid cessation of nucleolar RNA synthesis (ribosomal DNA transcription) and concluded that a rapidly turning over protein was required for RNA polymerase I (pol I)1 activity in vivo. Subsequent studies have demonstrated that transcription by RNA polymerase I is subject to regulation at many levels (4Hannan K.M. Hannan R.D. Rothblum L.I. Front. Biosci. 1998; 3: d376-d398Crossref PubMed Google Scholar, 5Grummt I. Prog. Nucleic Acids Res. Mol. Biol. 1999; 62: 109-154Crossref PubMed Scopus (204) Google Scholar). At least three, and possibly more, polymerase-associated proteins, TIF-IA, Factor C*, and TFIC (6Cavanaugh A.H. Gokal P.K. Lawther R.P. Thompson E.A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 718-721Crossref PubMed Scopus (33) Google Scholar, 7Buttgereit D. Pflugfelder G. Grummt I. Nucleic Acids Res. 1985; 13: 8165-8180Crossref PubMed Scopus (76) Google Scholar, 8Tower J. Sollner-Webb B. Cell. 1987; 50: 873-883Abstract Full Text PDF PubMed Scopus (70) Google Scholar), have been demonstrated to contribute to the regulation of rDNA transcription. TIF-IA and Factor C* were identified as factors that were required for the complementation of extracts of quiescent or cycloheximide-treated cells. TFIC was identified as that activity required to reconstitute transcription by extracts of glucocorticoid-treated P1798 cells. This lymphosarcoma cell line exits the cell cycle in response to the synthetic glucocorticoid dexamethasone (DEX) (6Cavanaugh A.H. Gokal P.K. Lawther R.P. Thompson E.A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 718-721Crossref PubMed" @default.
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• W1995771026 title "Rrn3 Phosphorylation Is a Regulatory Checkpoint for Ribosome Biogenesis" @default.
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