Proteomic Analysis Reveals a Role for the GTPase RPAP4/GPN1 and the Cochaperone RPAP3 in Biogenesis of All Three Nuclear RNA Polymerases

  • Diane Forget
  • Philippe Cloutier
  • Céline Domecq
  • Benoit Coulombe


Biogenesis of nuclear RNA polymerases (RNAP) is a poorly understood, yet central molecular process in eukaryotes. Recent analysis of interaction partners of RNAP II, the enzyme that synthesizes protein-coding mRNAs, in the soluble fraction of cell extracts identified a series of factors that play central roles in RNAP II biogenesis. The GPN loop GTPase RPAP4/GPN1 was shown to be required for nuclear import of RNAP II, and the HSP90 co-factor RPAP3 is essential for cytoplasmic assembly of this multisubunit enzyme. Examination of the list of interactors for RNAP II as well as RPAP4/GPN1 and RPAP3 reveals the presence of many specific subunits of RNAP I and III, which synthesize most of the cell’s non-coding transcripts. This finding suggests that biogenesis of all three nuclear RNAPs may be coupled. Silencing of RPAP4/GPN1 and RPAP3 further indicates that both factors are essential for normal nuclear localization of the three polymerases. We present a model in which biogenesis of RNAP I, II and III is integrated through the action of assembly and nuclear import factors.


RNA polymerase RNA polymerase II-associated proteins (RPAP) Biogenesis Nuclear import Assembly 



We wish to thank members of our laboratory for helpful discussions. This work was supported by grants from the Fonds de la recherche en santé du Québec (FRSQ) and the Canadian Institutes for Health Research (CIHR).


  1. 1.
    Conaway JW, Shilatifard A, Dvir A, Conaway RC. Control of elongation by RNA polymerase II. Trends Biochem Sci. 2000;25(8):375–80.PubMedCrossRefGoogle Scholar
  2. 2.
    Coulombe B, Burton ZF. DNA bending and wrapping around RNA polymerase: a “revolutionary” model describing transcriptional mechanisms. Microbiol Mol Biol Rev. 1999;63(2):457–78.PubMedGoogle Scholar
  3. 3.
    Hampsey M. Molecular genetics of the RNA polymerase II general transcriptional machinery. Microbiol Mol Biol Rev. 1998;62(2):465–503.PubMedGoogle Scholar
  4. 4.
    Kornberg RD. The molecular basis of eukaryotic transcription. Proc Natl Acad Sci USA. 2007;104(32):12955–61.PubMedCrossRefGoogle Scholar
  5. 5.
    Dey A, Nishiyama A, Karpova T, McNally J, Ozato K. Brd4 marks select genes on mitotic chromatin and directs postmitotic transcription. Mol Biol Cell. 2009;20(23):4899–909.PubMedCrossRefGoogle Scholar
  6. 6.
    Parsons GG, Spencer CA. Mitotic repression of RNA polymerase II transcription is accompanied by release of transcription elongation complexes. Mol Cell Biol. 1997;17(10):5791–802.PubMedGoogle Scholar
  7. 7.
    Tseng LC, Chen RH. Temporal control of nuclear envelope assembly by phosphorylation of lamin B receptor. Mol Biol Cell. 2011;22(18):3306–17.PubMedCrossRefGoogle Scholar
  8. 8.
    Czeko E, Seizl M, Augsberger C, Mielke T, Cramer P. Iwr1 directs RNA polymerase II nuclear import. Mol Cell. 2011;42(2):261–6.PubMedCrossRefGoogle Scholar
  9. 9.
    Jeronimo C, Forget D, Bouchard A, Li Q, Chua G, Poitras C, et al. Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme. Mol Cell. 2007;27(2):262–74.PubMedCrossRefGoogle Scholar
  10. 10.
    Gras S, Chaumont V, Fernandez B, Carpentier P, Charrier-Savournin F, Schmitt S, et al. Structural insights into a new homodimeric self-activated GTPase family. EMBO Rep. 2007;8(6):569–75.PubMedCrossRefGoogle Scholar
  11. 11.
    Carre C, Shiekhattar R. Human GTPases associate with RNA polymerase II to mediate its nuclear import. Mol Cell Biol. 2011;31(19):3953–62.PubMedCrossRefGoogle Scholar
  12. 12.
    Forget D, Lacombe AA, Cloutier P, Al Khoury R, Bouchard A, Lavallee-Adam M, et al. 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. 2010;9(12):2827–39.PubMedCrossRefGoogle Scholar
  13. 13.
    Reyes-Pardo H, Barbosa-Camacho AA, Perez-Mejia AE, Lara-Chacon B, Salas-Estrada LA, Robledo-Rivera AY, et al. A nuclear export sequence in GPN-loop GTPase 1, an essential protein for nuclear targeting of RNA polymerase II, is necessary and sufficient for nuclear export. Biochim Biophys Acta. 2012;1823(10):1756–66.PubMedCrossRefGoogle Scholar
  14. 14.
    Cloutier P, Al Khoury R, Lavallee-Adam M, Faubert D, Jiang H, Poitras C, et al. High-resolution mapping of the protein interaction network for the human transcription machinery and affinity purification of RNA polymerase II-associated complexes. Methods. 2009;48(4):381–6.PubMedCrossRefGoogle Scholar
  15. 15.
    Gstaiger M, Luke B, Hess D, Oakeley EJ, Wirbelauer C, Blondel M, et al. Control of nutrient-sensitive transcription programs by the unconventional prefoldin URI. Science. 2003;302(5648):1208–12.PubMedCrossRefGoogle Scholar
  16. 16.
    Sardiu ME, Cai Y, Jin J, Swanson SK, Conaway RC, Conaway JW, et al. Probabilistic assembly of human protein interaction networks from label-free quantitative proteomics. Proc Natl Acad Sci USA. 2008;105(5):1454–9.PubMedCrossRefGoogle Scholar
  17. 17.
    Martin-Benito J, Gomez-Reino J, Stirling PC, Lundin VF, Gomez-Puertas P, Boskovic J, et al. Divergent substrate-binding mechanisms reveal an evolutionary specialization of eukaryotic prefoldin compared to its archaeal counterpart. Structure. 2007;15(1):101–10.PubMedCrossRefGoogle Scholar
  18. 18.
    Simons CT, Staes A, Rommelaere H, Ampe C, Lewis SA, Cowan NJ. Selective contribution of eukaryotic prefoldin subunits to actin and tubulin binding. J Biol Chem. 2004;279(6):4193–203.Google Scholar
  19. 19.
    McKeegan KS, Debieux CM, Watkins NJ. Evidence that the AAA+ proteins TIP48 and TIP49 bridge interactions between 15.5K and the related NOP56 and NOP58 proteins during box C/D snoRNP biogenesis. Mol Cell Biol. 2009;29(18):4971–81.PubMedCrossRefGoogle Scholar
  20. 20.
    Boulon S, Pradet-Balade B, Verheggen C, Molle D, Boireau S, Georgieva M, et al. HSP90 and its R2TP/Prefoldin-like cochaperone are involved in the cytoplasmic assembly of RNA polymerase II. Mol Cell. 2010;39(6):912–24.PubMedCrossRefGoogle Scholar
  21. 21.
    Dundr M, Hoffmann-Rohrer U, Hu Q, Grummt I, Rothblum LI, Phair RD, et al. A kinetic framework for a mammalian RNA polymerase in vivo. Science. 2002;298(5598):1623–6.PubMedCrossRefGoogle Scholar
  22. 22.
    Hardeland U, Hurt E. Coordinated nuclear import of RNA polymerase III subunits. Traffic. 2006;7(4):465–73.PubMedCrossRefGoogle Scholar
  23. 23.
    Wild T, Cramer P. Biogenesis of multisubunit RNA polymerases. Trends Biochem Sci. 2012;37(3):99–105.PubMedCrossRefGoogle Scholar
  24. 24.
    Archambault J, Friesen JD. Genetics of eukaryotic RNA polymerases I, II, and III. Microbiol Rev. 1993;57(3):703–24.PubMedGoogle Scholar
  25. 25.
    Young RA. RNA polymerase II. Annu Rev Biochem. 1991;60:689–715.PubMedCrossRefGoogle Scholar
  26. 26.
    Gingras AC, Gstaiger M, Raught B, Aebersold R. Analysis of protein complexes using mass spectrometry. Nat Rev Mol Cell Biol. 2007;8(8):645–54.PubMedCrossRefGoogle Scholar
  27. 27.
    Jeronimo C, Langelier MF, Zeghouf M, Cojocaru M, Bergeron D, Baali D, et al. RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits. Mol Cell Biol. 2004;24(16):7043–58.PubMedCrossRefGoogle Scholar
  28. 28.
    Lavallee-Adam M, Rousseau J, Domecq C, Bouchard A, Forget D, Faubert D, et al. Discovery of cell compartment specific protein-protein interactions using affinity purification combined with tandem mass spectrometry. J Proteome Res. 2013;12(1):272–81.PubMedCrossRefGoogle Scholar
  29. 29.
    Baillat D, Hakimi MA, Naar AM, Shilatifard A, Cooch N, Shiekhattar R. Integrator, a multiprotein mediator of small nuclear RNA processing, associates with the C-terminal repeat of RNA polymerase II. Cell. 2005;123(2):265–76.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Diane Forget
    • 1
  • Philippe Cloutier
    • 1
  • Céline Domecq
    • 1
  • Benoit Coulombe
    • 1
  1. 1.Laboratory of Gene Transcription and ProteomicsInstitut de recherches cliniques de MontréalMontréalCanada

Personalised recommendations