All Title Author
Keywords Abstract

PLOS Genetics  2013 

The Prefoldin Bud27 Mediates the Assembly of the Eukaryotic RNA Polymerases in an Rpb5-Dependent Manner

DOI: 10.1371/journal.pgen.1003297

Full-Text   Cite this paper   Add to My Lib


The unconventional prefoldin URI/RMP, in humans, and its orthologue in yeast, Bud27, have been proposed to participate in the biogenesis of the RNA polymerases. However, this role of Bud27 has not been confirmed and is poorly elucidated. Our data help clarify the mechanisms governing biogenesis of the three eukaryotic RNA pols. We show evidence that Bud27 is the first example of a protein that participates in the biogenesis of the three eukaryotic RNA polymerases and the first example of a protein modulating their assembly instead of their nuclear transport. In addition we demonstrate that the role of Bud27 in RNA pols biogenesis depends on Rpb5. In fact, lack of BUD27 affects growth and leads to a substantial accumulation of the three RNA polymerases in the cytoplasm, defects offset by the overexpression of RPB5. Supporting this, our data demonstrate that the lack of Bud27 affects the correct assembly of Rpb5 and Rpb6 to the three RNA polymerases, suggesting that this process occurs in the cytoplasm and is a required step prior to nuclear import. Also, our data support the view that Rpb5 and Rpb6 assemble somewhat later than the rest of the complexes. Furthermore, Bud27 Rpb5-binding but not PFD-binding domain is necessary for RNA polymerases biogenesis. In agreement, we also demonstrate genetic interactions between BUD27, RPB5, and RPB6. Bud27 shuttles between the nucleus and the cytoplasm in an Xpo1-independent manner, and also independently of microtubule polarization and possibly independently of its association with the RNA pols. Our data also suggest that the role of Bud27 in RNA pols biogenesis is independent of the chaperone prefoldin (PFD) complex and of Iwr1. Finally, the role of URI seems to be conserved in humans, suggesting conserved mechanisms in RNA pols biogenesis.


[1]  Werner F, Grohmann D (2011) Evolution of multisubunit RNA polymerases in the three domains of life. Nat Rev Microbiol 9: 85–98. doi: 10.1038/nrmicro2507
[2]  Zaros C, Briand JF, Boulard Y, Labarre-Mariotte S, Garcia-Lopez MC, et al. (2007) Functional organization of the Rpb5 subunit shared by the three yeast RNA polymerases. Nucleic Acids Res 35: 634–647. doi: 10.1093/nar/gkl686
[3]  Werner M, Thuriaux P, Soutourina J (2009) Structure-function analysis of RNA polymerases I and III. Curr Opin Struct Biol 19: 740–745. doi: 10.1016/
[4]  Cramer P, Armache KJ, Baumli S, Benkert S, Brueckner F, et al. (2008) Structure of eukaryotic RNA polymerases. Annu Rev Biophys 37: 337–352. doi: 10.1146/annurev.biophys.37.032807.130008
[5]  Fernandez-Tornero C, Bottcher B, Riva M, Carles C, Steuerwald U, et al. (2007) Insights into transcription initiation and termination from the electron microscopy structure of yeast RNA polymerase III. Mol Cell 25: 813–823. doi: 10.1016/j.molcel.2007.02.016
[6]  Czeko E, Seizl M, Augsberger C, Mielke T, Cramer P (2011) Iwr1 Directs RNA Polymerase II Nuclear Import. Mol Cell 42: 261–266. doi: 10.1016/j.molcel.2011.02.033
[7]  Staresincic L, Walker J, Dirac-Svejstrup AB, Mitter R, Svejstrup JQ (2011) GTP-dependent binding and nuclear transport of RNA polymerase II by NPA3. J Biol Chem doi: 10.1074/jbc.m111.286161
[8]  Forget D, Lacombe AA, Cloutier P, Al-Khoury R, Bouchard A, et al. (2010) The protein interaction network of the human transcription machinery reveals a role for the conserved GTPase RPAP4/GPN1 and microtubule assembly in nuclear import and biogenesis of RNA polymerase II. Mol Cell Proteomics 9: 2827–2839. doi: 10.1074/mcp.m110.003616
[9]  Carre C, Shiekhattar R (2011) Human GTPases Associate with RNA Polymerase II To Mediate Its Nuclear Import. Mol Cell Biol 31: 3953–3962. doi: 10.1128/mcb.05442-11
[10]  Wild T, Cramer P (2012) Biogenesis of multisubunit RNA polymerases. Trends Biochem Sci 37: 99–105. doi: 10.1016/j.tibs.2011.12.001
[11]  Egloff S, Zaborowska J, Laitem C, Kiss T, Murphy S (2012) Ser7 phosphorylation of the CTD recruits the RPAP2 Ser5 phosphatase to snRNA genes. Mol Cell 45: 111–122. doi: 10.1016/j.molcel.2011.11.006
[12]  Boulon S, Pradet-Balade B, Verheggen C, Molle D, Boireau S, et al. (2010) HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II. Mol Cell 39: 912–924. doi: 10.1016/j.molcel.2010.08.023
[13]  Cloutier P, Coulombe B (2010) New insights into the biogenesis of nuclear RNA polymerases? Biochem Cell Biol 88: 211–221. doi: 10.1139/o09-173
[14]  Dorjsuren D, Lin Y, Wei W, Yamashita T, Nomura T, et al. (1998) RMP, a novel RNA polymerase II subunit 5-interacting protein, counteracts transactivation by hepatitis B virus X protein. Mol Cell Biol 18: 7546–7555.
[15]  Le TT, Zhang S, Hayashi N, Yasukawa M, Delgermaa L, et al. (2005) Mutational analysis of human RNA polymerase II subunit 5 (RPB5): the residues critical for interactions with TFIIF subunit RAP30 and hepatitis B virus X protein. J Biochem (Tokyo) 138: 215–224. doi: 10.1093/jb/mvi119
[16]  Wei W, Gu JX, Zhu CQ, Sun FY, Dorjsuren D, et al. (2003) Interaction with general transcription factor IIF (TFIIF) is required for the suppression of activated transcription by RPB5-mediating protein (RMP). Cell Res 13: 111–120. doi: 10.1038/
[17]  Yart A, Gstaiger M, Wirbelauer C, Pecnik M, Anastasiou D, et al. (2005) The HRPT2 tumor suppressor gene product parafibromin associates with human PAF1 and RNA polymerase II. Mol Cell Biol 25: 5052–5060. doi: 10.1128/mcb.25.12.5052-5060.2005
[18]  Gstaiger M, Luke B, Hess D, Oakeley EJ, Wirbelauer C, et al. (2003) Control of nutrient-sensitive transcription programs by the unconventional prefoldin URI. Science 302: 1208–1212. doi: 10.1126/science.1088401
[19]  Deplazes A, Mockli N, Luke B, Auerbach D, Peter M (2009) Yeast Uri1p promotes translation initiation and may provide a link to cotranslational quality control. EMBO J 28: 1429–1441. doi: 10.1038/emboj.2009.98
[20]  Mita P, Savas JN, Djouder N, Yates JR 3rd, Ha S, et al. (2011) REGULATION OF ANDROGEN RECEPTOR MEDIATED TRANSCRIPTION BY RPB5 BINDING PROTEIN URI/RMP. Mol Cell Biol doi: 10.1128/mcb.05429-11
[21]  Kirchner J, Vissi E, Gross S, Szoor B, Rudenko A, et al. (2008) Drosophila Uri, a PP1alpha binding protein, is essential for viability, maintenance of DNA integrity and normal transcriptional activity. BMC Mol Biol 9: 36. doi: 10.1186/1471-2199-9-36
[22]  Delgermaa L, Hayashi N, Dorjsuren D, Nomura T, Thuy le TT, et al. (2004) Subcellular localization of RPB5-mediating protein and its putative functional partner. Mol Cell Biol 24: 8556–8566. doi: 10.1128/mcb.24.19.8556-8566.2004
[23]  Mockli N, Deplazes A, Hassa PO, Zhang Z, Peter M, et al. (2007) Yeast split-ubiquitin-based cytosolic screening system to detect interactions between transcriptionally active proteins. Biotechniques 42: 725–730. doi: 10.2144/000112455
[24]  Krogan NJ, Cagney G, Yu H, Zhong G, Guo X, et al. (2006) Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440: 637–643. doi: 10.1038/nature04670
[25]  Gari E, Piedrafita L, Aldea M, Herrero E (1997) A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae. Yeast 13: 837–848.
[26]  Geissler S, Siegers K, Schiebel E (1998) A novel protein complex promoting formation of functional alpha- and gamma-tubulin. EMBO J 17: 952–966. doi: 10.1093/emboj/17.4.952
[27]  Lopez N, Halladay J, Walter W, Craig EA (1999) SSB, encoding a ribosome-associated chaperone, is coordinately regulated with ribosomal protein genes. J Bacteriol 181: 3136–3143.
[28]  Kosugi S, Hasebe M, Tomita M, Yanagawa H (2009) Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs. Proc Natl Acad Sci U S A 106: 10171–10176. doi: 10.1073/pnas.0900604106
[29]  la Cour T, Kiemer L, Molgaard A, Gupta R, Skriver K, et al. (2004) Analysis and prediction of leucine-rich nuclear export signals. Protein Eng Des Sel 17: 527–536. doi: 10.1093/protein/gzh062
[30]  Peiro-Chova L, Estruch F (2009) The yeast RNA polymerase II-associated factor Iwr1p is involved in the basal and regulated transcription of specific genes. J Biol Chem 284: 28958–28967. doi: 10.1074/jbc.m109.012153
[31]  Corden J (2011) Going nuclear: transcribers in transit. Mol Cell 42: 143–145. doi: 10.1016/j.molcel.2011.04.001
[32]  Mosley AL, Sardiu ME, Pattenden SG, Workman JL, Florens L, et al. (2011) Highly reproducible label free quantitative proteomic analysis of RNA polymerase complexes. Mol Cell Proteomics 10: M110 000687. doi: 10.1074/mcp.m110.000687
[33]  Lotan R, Bar-On VG, Harel-Sharvit L, Duek L, Melamed D, et al. (2005) The RNA polymerase II subunit Rpb4p mediates decay of a specific class of mRNAs. Genes Dev 19: 3004–3016. doi: 10.1101/gad.353205
[34]  Sole C, Nadal-Ribelles M, Kraft C, Peter M, Posas F, et al. (2011) Control of Ubp3 ubiquitin protease activity by the Hog1 SAPK modulates transcription upon osmostress. EMBO J 30: 3274–3284. doi: 10.1038/emboj.2011.227
[35]  Graumann J, Dunipace LA, Seol JH, McDonald WH, Yates JR 3rd, et al. (2004) Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast. Mol Cell Proteomics 3: 226–237. doi: 10.1074/mcp.m300099-mcp200
[36]  Miyazawa M, Tashiro E, Kitaura H, Maita H, Suto H, et al. (2011) Prefoldin subunits are protected from ubiquitin-proteasome system-mediated degradation by forming complex with other constituent subunits. J Biol Chem 286: 19191–19203. doi: 10.1074/jbc.m110.216259
[37]  Collins SR, Miller KM, Maas NL, Roguev A, Fillingham J, et al. (2007) Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map. Nature 446: 806–810. doi: 10.1038/nature05649
[38]  Wilmes GM, Bergkessel M, Bandyopadhyay S, Shales M, Braberg H, et al. (2008) A genetic interaction map of RNA-processing factors reveals links between Sem1/Dss1-containing complexes and mRNA export and splicing. Mol Cell 32: 735–746. doi: 10.1016/j.molcel.2008.11.012
[39]  Ye P, Peyser BD, Pan X, Boeke JD, Spencer FA, et al. (2005) Gene function prediction from congruent synthetic lethal interactions in yeast. Mol Syst Biol 1: 2005 0026. doi: 10.1038/msb4100034
[40]  Zheng J, Benschop JJ, Shales M, Kemmeren P, Greenblatt J, et al. (2010) Epistatic relationships reveal the functional organization of yeast transcription factors. Mol Syst Biol 6: 420. doi: 10.1038/msb.2010.77
[41]  Costanzo M, Baryshnikova A, Bellay J, Kim Y, Spear ED, et al. (2010) The genetic landscape of a cell. Science 327: 425–431. doi: 10.1126/science.1180823
[42]  Esberg A, Moqtaderi Z, Fan X, Lu J, Struhl K, et al. (2011) Iwr1 protein is important for preinitiation complex formation by all three nuclear RNA polymerases in Saccharomyces cerevisiae. PLoS ONE 6: e20829 doi:10.1371/journal.pone.0020829.
[43]  Boulon S, Bertrand E, Pradet-Balade B (2012) HSP90 and the R2TP co-chaperone complex: Building multi-protein machineries essential for cell growth and gene expression. RNA Biol 9. doi: 10.4161/rna.18494
[44]  Koh JL, Ding H, Costanzo M, Baryshnikova A, Toufighi K, et al. (2010) DRYGIN: a database of quantitative genetic interaction networks in yeast. Nucleic Acids Res 38: D502–507. doi: 10.1093/nar/gkp820
[45]  Cramer P, Bushnell DA, Kornberg RD (2001) Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution. Science 292: 1863–1876. doi: 10.1126/science.1059493
[46]  Garcia-Lopez MC, Pelechano V, Miron-Garcia MC, Garrido-Godino AI, Garcia A, et al. (2011) The conserved foot domain of RNA pol II associates with proteins involved in transcriptional initiation and/or early elongation. Genetics 189: 1235–1248. doi: 10.1534/genetics.111.133215
[47]  Longtine MS, McKenzie A 3rd, Demarini DJ, Shah NG, Wach A, et al. (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14: 953–961.
[48]  Sheff MA, Thorn KS (2004) Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae. Yeast 21: 661–670.
[49]  Soutourina J, Bordas-Le Floch V, Gendrel G, Flores A, Ducrot C, et al. (2006) Rsc4 connects the chromatin remodeler RSC to RNA polymerases. Mol Cell Biol 26: 4920–4933. doi: 10.1128/mcb.00415-06
[50]  Rodriguez-Navarro S, Fischer T, Luo MJ, Antunez O, Brettschneider S, et al. (2004) Sus1, a functional component of the SAGA histone acetylase complex and the nuclear pore-associated mRNA export machinery. Cell 116: 75–86. doi: 10.1016/s0092-8674(03)01025-0
[51]  Liang C, Stillman B (1997) Persistent initiation of DNA replication and chromatin-bound MCM proteins during the cell cycle in cdc6 mutants. Genes Dev 11: 3375–3386. doi: 10.1101/gad.11.24.3375
[52]  Thomas BJ, Rothstein R (1989) Elevated recombination rates in transcriptionally active DNA. Cell 56: 619–630. doi: 10.1016/0092-8674(89)90584-9
[53]  Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122: 19–27.
[54]  Stade K, Ford CS, Guthrie C, Weis K (1997) Exportin 1 (Crm1p) is an essential nuclear export factor. Cell 90: 1041–1050. doi: 10.1016/s0092-8674(00)80370-0
[55]  Van Mullem V, Wery M, Werner M, Vandenhaute J, Thuriaux P (2002) The Rpb9 subunit of RNA polymerase II binds transcription factor TFIIE and interferes with the SAGA and elongator histone acetyltransferases. J Biol Chem 277: 10220–10225. doi: 10.1074/jbc.m107207200
[56]  Bonneaud N, Ozier-Kalogeropoulos O, Li GY, Labouesse M, Minvielle-Sebastia L, et al. (1991) A family of low and high copy replicative, integrative and single- stranded S. cerevisiae/E. coli shuttle vectors. Yeast 7: 609–615.
[57]  Rubbi L, Labarre-Mariotte S, Chedin S, Thuriaux P (1999) Functional characterization of ABC10alpha, an essential polypeptide shared by all three forms of eukaryotic DNA-dependent RNA polymerases. J Biol Chem 274: 31485–31492. doi: 10.1074/jbc.274.44.31485


comments powered by Disqus