Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

RNA polymerase II termination involves C-terminal-domain tyrosine dephosphorylation by CPF subunit Glc7

Nature Structural & Molecular Biology volume 21, pages 175–179 (2014)Cite this article

Subjects

Abstract

At the 3′ ends of protein-coding genes, RNA polymerase (Pol) II is dephosphorylated at tyrosine residues (Tyr1) of its C-terminal domain (CTD). In addition, the associated cleavage-and-polyadenylation factor (CPF) cleaves the transcript and adds a poly(a) tail. Whether these events are coordinated and how they lead to transcription termination remains poorly understood. Here we show that CPF from Saccharomyces cerevisiae is a Pol II–CTD phosphatase and that the CPF subunit Glc7 dephosphorylates Tyr1 in vitro. In vivo, the activity of Glc7 is required for normal Tyr1 dephosphorylation at the polyadenylation site, for recruitment of termination factors Pcf11 and Rtt103 and for normal Pol II termination. These results show that transcription termination involves Tyr1 dephosphorylation of the CTD and indicate that pre-mRNA processing by CPF and transcription termination are coupled via Glc7-dependent Pol II–Tyr1 dephosphorylation.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: CPF subunit Glc7 is a Pol II–CTD Tyr1 phosphatase in vitro.
Figure 2: Glc7 is required for dephosphorylation of Tyr1 but not Ser2 in vivo.
Figure 3: Ssu72 does not change Tyr1 phosphorylation levels in vitro and in vivo.
Figure 4: Tyr1 dephosphorylation by Glc7 is required for normal termination-factor recruitment and transcription termination in vivo.

Similar content being viewed by others

Accession codes

Primary accessions

ArrayExpress

References

  1. Brannan, K. & Bentley, D.L. Control of transcriptional elongation by RNA Polymerase II: a retrospective. Genet. Res. Int. 2012, 170–173 (2012).

    Google Scholar 

  2. Buratowski, S. Progression through the RNA polymerase II CTD cycle. Mol. Cell 36, 541–546 (2009).

    Article  CAS  Google Scholar 

  3. Corden, J.L. Transcription: seven ups the code. Science 318, 1735–1736 (2007).

    Article  CAS  Google Scholar 

  4. Jeronimo, C., Bataille, A.R. & Robert, F. The writers, readers, and functions of the RNA Polymerase II C-terminal domain code. Chem. Rev. 113, 8491–8522 (2013).

    Article  CAS  Google Scholar 

  5. Komarnitsky, P., Cho, E.J. & Buratowski, S. Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. Genes Dev. 14, 2452–2460 (2000).

    Article  CAS  Google Scholar 

  6. Schroeder, S.C., Schwer, B., Shuman, S. & Bentley, D. Dynamic association of capping enzymes with transcribing RNA polymerase II. Genes Dev. 14, 2435–2440 (2000).

    Article  CAS  Google Scholar 

  7. Ahn, S.H., Kim, M. & Buratowski, S. Phosphorylation of serine 2 within the RNA polymerase II C-terminal domain couples transcription and 3′ end processing. Mol. Cell 13, 67–76 (2004).

    Article  CAS  Google Scholar 

  8. Mayer, A. et al. CTD tyrosine phosphorylation impairs termination factor recruitment to RNA polymerase II. Science 336, 1723–1725 (2012).

    Article  CAS  Google Scholar 

  9. Niño, C.A., Hérissant, L., Babour, A. & Dargemont, C. mRNA nuclear export in yeast. Chem. Rev. 113, 8523–8545 (2013).

    Article  Google Scholar 

  10. Garneau, N.L., Wilusz, J. & Wilusz, C.J. The highways and byways of mRNA decay. Nat. Rev. Mol. Cell Biol. 8, 113–126 (2007).

    Article  CAS  Google Scholar 

  11. Kapp, L.D. & Lorsch, J.R. The molecular mechanics of eukaryotic translation. Annu. Rev. Biochem. 73, 657–704 (2004).

    Article  CAS  Google Scholar 

  12. Zhao, J., Hyman, L. & Moore, C. Formation of mRNA 3′ ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mRNA synthesis. Microbiol. Mol. Biol. Rev. 63, 405–445 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Mandel, C.R., Bai, Y. & Tong, L. Protein factors in pre-mRNA 3′-end processing. Cell Mol. Life Sci. 65, 1099–1122 (2008).

    Article  CAS  Google Scholar 

  14. McCracken, S. et al. The C-terminal domain of RNA polymerase II couples mRNA processing to transcription. Nature 385, 357–361 (1997).

    Article  CAS  Google Scholar 

  15. Licatalosi, D.D. et al. Functional interaction of yeast pre-mRNA 3′ end processing factors with RNA polymerase II. Mol. Cell 9, 1101–1111 (2002).

    Article  CAS  Google Scholar 

  16. Hirose, Y. & Manley, J.L. RNA polymerase II is an essential mRNA polyadenylation factor. Nature 395, 93–96 (1998).

    Article  CAS  Google Scholar 

  17. Proudfoot, N.J. Ending the message: poly(a) signals then and now. Genes Dev. 25, 1770–1782 (2011).

    Article  CAS  Google Scholar 

  18. Birse, C.E., Minvielle-Sebastia, L., Lee, B.A. & Keller, W. Coupling termination of transcription to messenger RNA maturation in yeast. Science 280, 298–301 (1998).

    Article  CAS  Google Scholar 

  19. Barillà, D., Lee, B.A. & Proudfoot, N.J. Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 98, 445–450 (2001).

    PubMed  Google Scholar 

  20. Sadowski, M., Dichtl, B., Hübner, W. & Keller, W. Independent functions of yeast Pcf11p in pre-mRNA 3′ end processing and in transcription termination. EMBO J. 22, 2167–2177 (2003).

    Article  CAS  Google Scholar 

  21. Meinhart, A. & Cramer, P. Recognition of RNA polymerase II carboxy-terminal domain by 3′-RNA-processing factors. Nature 430, 223–226 (2004).

    Article  CAS  Google Scholar 

  22. Krishnamurthy, S., He, X., Reyes-Reyes, M., Moore, C. & Hampsey, M. Ssu72 is an RNA polymerase II CTD phosphatase. Mol. Cell 14, 387–394 (2004).

    Article  CAS  Google Scholar 

  23. Meinhart, A., Silberzahn, T. & Cramer, P. The mRNA transcription/processing factor Ssu72 is a potential tyrosine phosphatase. J. Biol. Chem. 278, 15917–15921 (2003).

    Article  CAS  Google Scholar 

  24. Nedea, E. et al. Organization and function of APT, a subcomplex of the yeast cleavage and polyadenylation factor involved in the formation of mRNA and small nucleolar RNA 3′-ends. J. Biol. Chem. 278, 33000–33010 (2003).

    Article  CAS  Google Scholar 

  25. Bataille, A.R. et al. A universal RNA polymerase II CTD cycle is orchestrated by complex interplays between kinase, phosphatase, and isomerase enzymes along genes. Mol. Cell 45, 158–170 (2012).

    Article  CAS  Google Scholar 

  26. Xiang, K. et al. Crystal structure of the human symplekin-Ssu72-CTD phosphopeptide complex. Nature 467, 729–733 (2010).

    Article  CAS  Google Scholar 

  27. Nedea, E. et al. The Glc7 phosphatase subunit of the cleavage and polyadenylation factor is essential for transcription termination on snoRNA genes. Mol. Cell 29, 577–587 (2008).

    Article  CAS  Google Scholar 

  28. Gilbert, W. & Guthrie, C. The Glc7p nuclear phosphatase promotes mRNA export by facilitating association of Mex67p with mRNA. Mol. Cell 13, 201–212 (2004).

    Article  CAS  Google Scholar 

  29. He, X. & Moore, C. Regulation of yeast mRNA 3′ end processing by phosphorylation. Mol. Cell 19, 619–629 (2005).

    Article  CAS  Google Scholar 

  30. Shi, Y. Serine/threonine phosphatases: mechanism through structure. Cell 139, 468–484 (2009).

    Article  CAS  Google Scholar 

  31. Chu, Y., Lee, E.Y. & Schlender, K.K. Activation of protein phosphatase 1: formation of a metalloenzyme. J. Biol. Chem. 271, 2574–2577 (1996).

    Article  CAS  Google Scholar 

  32. Egloff, M.P., Cohen, P.T., Reinemer, P. & Barford, D. Crystal structure of the catalytic subunit of human protein phosphatase 1 and its complex with tungstate. J. Mol. Biol. 254, 942–959 (1995).

    Article  CAS  Google Scholar 

  33. Haruki, H., Nishikawa, J. & Laemmli, U.K. The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes. Mol. Cell 31, 925–932 (2008).

    Article  CAS  Google Scholar 

  34. Mayer, A. et al. Uniform transitions of the general RNA polymerase II transcription complex. Nat. Struct. Mol. Biol. 17, 1272–1278 (2010).

    Article  CAS  Google Scholar 

  35. Cho, E.J., Kobor, M.S., Kim, M., Greenblatt, J. & Buratowski, S. Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of the RNA polymerase II C-terminal domain. Genes Dev. 15, 3319–3329 (2001).

    Article  CAS  Google Scholar 

  36. Peti, W., Nairn, A.C. & Page, R. Structural basis for protein phosphatase 1 regulation and specificity. FEBS J. 280, 596–611 (2013).

    Article  CAS  Google Scholar 

  37. MacKintosh, C. et al. Further evidence that inhibitor-2 acts like a chaperone to fold PP1 into its native conformation. FEBS Lett. 397, 235–238 (1996).

    Article  CAS  Google Scholar 

  38. Farkas, I., Dombrádi, V., Miskei, M., Szabados, L. & Koncz, C. Arabidopsis PPP family of serine/threonine phosphatases. Trends Plant Sci. 12, 169–176 (2007).

    Article  CAS  Google Scholar 

  39. Kim, T.-W. et al. Brassinosteroid signal transduction from cell-surface receptor kinases to nuclear transcription factors. Nat. Cell Biol. 11, 1254–1260 (2009).

    Article  CAS  Google Scholar 

  40. Chao, Y. et al. Structure and mechanism of the phosphotyrosyl phosphatase activator. Mol. Cell 23, 535–546 (2006).

    Article  CAS  Google Scholar 

  41. Kim, M. et al. The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II. Nature 432, 517–522 (2004).

    Article  CAS  Google Scholar 

  42. Logan, J., Falck-Pedersen, E., Darnell, J.E. & Shenk, T. A poly(a) addition site and a downstream termination region are required for efficient cessation of transcription by RNA polymerase II in the mouse βmaj-globin gene. Proc. Natl. Acad. Sci. USA 84, 8306–8310 (1987).

    Article  CAS  Google Scholar 

  43. Mischo, H.E. & Proudfoot, N.J. Disengaging polymerase: terminating RNA polymerase II transcription in budding yeast. Biochim. Biophys. Acta 1829, 174–185 (2013).

    Article  CAS  Google Scholar 

  44. Passmore, L.A. et al. Doc1 mediates the activity of the anaphase-promoting complex by contributing to substrate recognition. EMBO J. 22, 786–796 (2003).

    Article  CAS  Google Scholar 

  45. Sydow, J.F. et al. Structural basis of transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA. Mol. Cell 34, 710–721 (2009).

    Article  CAS  Google Scholar 

  46. Chapman, R.D. et al. Transcribing RNA polymerase II is phosphorylated at CTD residue serine-7. Science 318, 1780–1782 (2007).

    Article  CAS  Google Scholar 

  47. Mayer, A. et al. The spt5 C-terminal region recruits yeast 3′ RNA cleavage factor I. Mol. Cell Biol. 32, 1321–1331 (2012).

    Article  CAS  Google Scholar 

  48. Nagalakshmi, U. et al. The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 320, 1344–1349 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank A. Cheung (Cramer laboratory), N.A. Yewdall (Passmore laboratory) and the mass spectrometry facility at the MRC Laboratory of Molecular Biology (LMB) for help, D. Barford for discussions, E. Kremmer and D. Eick (Helmholtz Zentrum München) for antibodies and S. Munro (MRC-LMB) for yeast strains. A.D.E. was supported by a Woolf Fisher Trust Scholarship, and K.W. was supported by a fellowship from the Deutsche Forschungsgemeinschaft. Work in the lab of L.A.P. is supported by MRC grant MC_U105192715 (L.A.P.) and European Research Council (ERC) Starting Grant 261151 (L.A.P.). P.C. was supported by the Deutsche Forschungsgemeinschaft (SFB646, SFB960, SFB1064, CIPSM, NIM, QBM), an ERC Advanced Grant, the Jung-Stiftung and the Vallee Foundation.

Author information

Author notes

  1. Amelie Schreieck and Ashley D Easter: These authors contributed equally to this work.

Authors and Affiliations

  1. Gene Center, Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, Munich, Germany

    Amelie Schreieck, Stefanie Etzold, Michael Lidschreiber & Patrick Cramer

  2. Department of Biochemistry, CIPSM, Ludwig-Maximilians-Universität München, Munich, Germany

    Amelie Schreieck, Stefanie Etzold, Michael Lidschreiber & Patrick Cramer

  3. Medical Research Council (MRC) Laboratory of Molecular Biology, Cambridge, UK

    Ashley D Easter, Katrin Wiederhold & Lori A Passmore

  4. Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Patrick Cramer

Authors
  1. Amelie Schreieck
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ashley D Easter
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stefanie Etzold
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Katrin Wiederhold
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Michael Lidschreiber
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Patrick Cramer
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lori A Passmore
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.S., A.D.E., S.E. and K.W. performed experiments. M.L. analyzed data. L.A.P. and P.C. designed and supervised research. A.S., A.D.E., L.A.P. and P.C. wrote the manuscript.

Corresponding authors

Correspondence to Patrick Cramer or Lori A Passmore.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Growth analysis and fluorescence microscopy of anchor-away yeast strains.

(a) Serial dilutions of wild-type and Glc7 and Ssu72 anchor-away yeast strains plated on YPD (left panel) and YPD + rapamycin (right panel) show that rapamycin is lethal for the anchor-away strains but it has no effect on wild-type growth. FRB, FKBP12-rapamycin-binding. (b) Fluorescence microscopy of fixed cells of the Ssu72-FRB/Glc7-mCherry strain shows that Glc7 is located in both cytoplasm and nucleus (left panel). This distribution does not change when rapamycin is added to the cells (lower panel). DAPI stain is shown as a control (right panel).

Supplementary Figure 2 Depletion of Glc7 from the nucleus leads to a defect in Tyr1-P dephosphorylation.

ChIP-chip occupancy profile of Tyr1-phosphorylated Pol II over 619 genes without and with rapamycin (solid and dotted line, respectively; see Fig. 2a for normalized profiles). The profiles include the region from 250 nucleotides upstream of the transcription start site (TSS) to 400 nucleotides downstream of the polyadenylation site (pA).

Supplementary Figure 3 Genome-wide ChIP occupancy of Tyr1-phosphorylated Pol II around the pA site is not influenced by rapamycin in wild-type yeast.

ChIP-chip occupancy profiling of Tyr1-phosphorylated Pol II over 619 genes aligned at the pA site (dashed line) and normalized against the corresponding Rpb3 profile without and with rapamycin (violet line and violet dotted line, respectively). The profile in a region from 400 nucleotides upstream to 400 nucleotides downstream of the polyA site is shown.

Supplementary Figure 4 Uncropped western blots of Pol II CTD in vitro assays.

(a) Uncropped blots of Figure 1b in the main text. (b) Uncropped blots of Figure 3a in the main text. See main text for details.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 (PDF 2473 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schreieck, A., Easter, A., Etzold, S. et al. RNA polymerase II termination involves C-terminal-domain tyrosine dephosphorylation by CPF subunit Glc7. Nat Struct Mol Biol 21, 175–179 (2014). https://doi.org/10.1038/nsmb.2753

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2753

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing