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The Nrd1–Nab3–Sen1 termination complex interacts with the Ser5-phosphorylated RNA polymerase II C-terminal domain

Nature Structural & Molecular Biology volume 15, pages 795–804 (2008)Cite this article

Abstract

RNA polymerase II (Pol II) in Saccharomyces cerevisiae can terminate transcription via several pathways. To study how a mechanism is chosen, we analyzed recruitment of Nrd1, which cooperates with Nab3 and Sen1 to terminate small nucleolar RNAs and other short RNAs. Budding yeast contains three C-terminal domain (CTD) interaction domain (CID) proteins, which bind the CTD of the Pol II largest subunit. Rtt103 and Pcf11 act in mRNA termination, and both preferentially interact with CTD phosphorylated at Ser2. The crystal structure of the Nrd1 CID shows a fold similar to that of Pcf11, but Nrd1 preferentially binds to CTD phosphorylated at Ser5, the form found proximal to promoters. This indicates why Nrd1 cross-links near 5′ ends of genes and why the Nrd1–Nab3–Sen1 termination pathway acts specifically at short Pol II–transcribed genes. Nrd1 recruitment to genes involves a combination of interactions with CTD and Nab3.

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Figure 1: Structure of the Nrd1 CID.
Figure 2: Nrd1 binds preferentially to CTD-Ser5P.
Figure 3: Structural models for a Nrd1-CTD interaction.
Figure 4: The CTD and Nab3 interaction domains of Nrd1 are both important for interaction with Pol II.
Figure 5: Efficient Nrd1 recruitment to 5′ ends of Pol II–transcribed genes requires both the CID and Nab3 interaction domain.
Figure 6: Effect of Nrd1 deletions on snoRNA termination and processing.

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References

  1. Buratowski, S. Connections between mRNA 3′ end processing and transcription termination. Curr. Opin. Cell Biol. 17, 257–261 (2005).

    Article  CAS  PubMed  Google Scholar 

  2. Li, X. & Manley, J.L. Cotranscriptional processes and their influence on genome stability. Genes Dev. 20, 1838–1847 (2006).

    Article  CAS  PubMed  Google Scholar 

  3. Buratowski, S. The CTD code. Nat. Struct. Biol. 10, 679–680 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. 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  PubMed  PubMed Central  Google Scholar 

  5. 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  PubMed  PubMed Central  Google Scholar 

  6. Hampsey, M. & Reinberg, D. Tails of intrigue: phosphorylation of RNA polymerase II mediates histone methylation. Cell 113, 429–432 (2003).

    Article  CAS  PubMed  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  PubMed  Google Scholar 

  8. Bird, G., Zorio, D.A. & Bentley, D.L. RNA polymerase II carboxy-terminal domain phosphorylation is required for cotranscriptional pre-mRNA splicing and 3′-end formation. Mol. Cell. Biol. 24, 8963–8969 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. 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  PubMed  PubMed Central  Google Scholar 

  10. 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  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  12. Noble, C.G. et al. Key features of the interaction between Pcf11 CID and RNA polymerase II CTD. Nat. Struct. Mol. Biol. 12, 144–151 (2005).

    Article  CAS  PubMed  Google Scholar 

  13. Kim, M. et al. Distinct pathways for snoRNA and mRNA termination. Mol. Cell 24, 723–734 (2006).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  15. West, S., Gromak, N. & Proudfoot, N.J. Human 5′ → 3′ exonuclease Xrn2 promotes transcription termination at co-transcriptional cleavage sites. Nature 432, 522–525 (2004).

    Article  CAS  PubMed  Google Scholar 

  16. Carroll, K.L., Pradhan, D.A., Granek, J.A., Clarke, N.D. & Corden, J.L. Identification of cis elements directing termination of yeast nonpolyadenylated snoRNA transcripts. Mol. Cell. Biol. 24, 6241–6252 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Steinmetz, E.J., Conrad, N.K., Brow, D.A. & Corden, J.L. RNA-binding protein Nrd1 directs poly(A)-independent 3′-end formation of RNA polymerase II transcripts. Nature 413, 327–331 (2001).

    Article  CAS  PubMed  Google Scholar 

  18. Conrad, N.K. et al. A yeast heterogeneous nuclear ribonucleoprotein complex associated with RNA polymerase II. Genetics 154, 557–571 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Steinmetz, E.J. & Brow, D.A. Repression of gene expression by an exogenous sequence element acting in concert with a heterogeneous nuclear ribonucleoprotein-like protein, Nrd1, and the putative helicase Sen1. Mol. Cell. Biol. 16, 6993–7003 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Steinmetz, E.J. & Brow, D.A. Control of pre-mRNA accumulation by the essential yeast protein Nrd1 requires high-affinity transcript binding and a domain implicated in RNA polymerase II association. Proc. Natl. Acad. Sci. USA 95, 6699–6704 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Vasiljeva, L. & Buratowski, S. Nrd1 interacts with the nuclear exosome for 3′ processing of RNA polymerase II transcripts. Mol. Cell 21, 239–248 (2006).

    Article  CAS  PubMed  Google Scholar 

  22. Houseley, J., LaCava, J. & Tollervey, D. RNA-quality control by the exosome. Nat. Rev. Mol. Cell Biol. 7, 529–539 (2006).

    Article  CAS  PubMed  Google Scholar 

  23. Steinmetz, E.J. et al. Genome-wide distribution of yeast RNA polymerase II and its control by Sen1 helicase. Mol. Cell 24, 735–746 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. David, L. et al. A high-resolution map of transcription in the yeast genome. Proc. Natl. Acad. Sci. USA 103, 5320–5325 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Davis, C.A. & Ares, M. Jr. Accumulation of unstable promoter-associated transcripts upon loss of the nuclear exosome subunit Rrp6p in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 103, 3262–3267 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Houalla, R. et al. Microarray detection of novel nuclear RNA substrates for the exosome. Yeast 23, 439–454 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Samanta, M.P., Tongprasit, W., Sethi, H., Chin, C.S. & Stolc, V. Global identification of noncoding RNAs in Saccharomyces cerevisiae by modulating an essential RNA processing pathway. Proc. Natl. Acad. Sci. USA 103, 4192–4197 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Arigo, J.T., Eyler, D.E., Carroll, K.L. & Corden, J.L. Termination of cryptic unstable transcripts is directed by yeast RNA-binding proteins Nrd1 and Nab3. Mol. Cell 23, 841–851 (2006).

    Article  CAS  PubMed  Google Scholar 

  29. Lykke-Andersen, S. & Jensen, T.H. CUT it out: silencing of noise in the transcriptome. Nat. Struct. Mol. Biol. 13, 860–861 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Thiebaut, M., Kisseleva-Romanova, E., Rougemaille, M., Boulay, J. & Libri, D. Transcription termination and nuclear degradation of cryptic unstable transcripts: a role for the Nrd1-Nab3 pathway in genome surveillance. Mol. Cell 23, 853–864 (2006).

    Article  CAS  PubMed  Google Scholar 

  31. Wyers, F. et al. Cryptic Pol II transcripts are degraded by a nuclear quality control pathway involving a new poly(A) polymerase. Cell 121, 725–737 (2005).

    Article  CAS  PubMed  Google Scholar 

  32. Vanacova, S. & Stef, R. The exosome and RNA quality control in the nucleus. EMBO Rep. 8, 651–657 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Meinhart, A., Kamenski, T., Hoeppner, S., Baumli, S. & Cramer, P. A structural perspective of CTD function. Genes Dev. 19, 1401–1415 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. Yuryev, A. et al. The C-terminal domain of the largest subunit of RNA polymerase II interacts with a novel set of serine/arginine-rich proteins. Proc. Natl. Acad. Sci. USA 93, 6975–6980 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Barilla, 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Kim, M., Ahn, S.H., Krogan, N.J., Greenblatt, J.F. & Buratowski, S. Transitions in RNA polymerase II elongation complexes at the 3′ ends of genes. EMBO J. 23, 354–364 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Sadowski, M., Dichtl, B., Hubner, 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  PubMed  PubMed Central  Google Scholar 

  38. Fabrega, C., Shen, V., Shuman, S. & Lima, C.D. Structure of an mRNA capping enzyme bound to the phosphorylated carboxy-terminal domain of RNA polymerase II. Mol. Cell 11, 1549–1561 (2003).

    Article  CAS  PubMed  Google Scholar 

  39. Verdecia, M.A., Bowman, M.E., Lu, K.P., Hunter, T. & Noel, J.P. Structural basis for phosphoserine-proline recognition by group IV WW domains. Nat. Struct. Biol. 7, 639–643 (2000).

    Article  CAS  PubMed  Google Scholar 

  40. Carroll, K.L., Ghirlando, R., Ames, J.M. & Corden, J.L. Interaction of yeast RNA-binding proteins Nrd1 and Nab3 with RNA polymerase II terminator elements. RNA 13, 361–373 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Arigo, J.T., Carroll, K.L., Ames, J.M. & Corden, J.L. Regulation of yeast NRD1 expression by premature transcription termination. Mol. Cell 21, 641–651 (2006).

    Article  CAS  PubMed  Google Scholar 

  42. Zhang, Z., Fu, J. & Gilmour, D.S. CTD-dependent dismantling of the RNA polymerase II elongation complex by the pre-mRNA 3′-end processing factor, Pcf11. Genes Dev. 19, 1572–1580 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 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  PubMed  Google Scholar 

  44. Steinmetz, E.J., Ng, S.B., Cloute, J.P. & Brow, D.A. Cis- and trans-acting determinants of transcription termination by yeast RNA polymerase II. Mol. Cell. Biol. 26, 2688–2696 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Gudipati, R.K., Villa, T., Boulay, J. & Libri, D. Phosphorylation of the RNA polymerase II C-terminal domain dictates transcription termination choice. Nat. Struct. Mol. Biol. advance online publication, doi:10.1038/nsmb.1460 (27 July 2008).

  46. Kumaki, Y., Matsushima, N., Yoshida, H., Nitta, K. & Hikichi, K. Structure of the YSPTSPS repeat containing two SPXX motifs in the CTD of RNA polymerase II: NMR studies of cyclic model peptides reveal that the SPTS turn is more stable than SPSY in water. Biochim. Biophys. Acta 1548, 81–93 (2001).

    Article  CAS  PubMed  Google Scholar 

  47. Becker, R., Loll, B. & Meinhart, A. Snapshots of the RNA processing factor SCAF8 bound to different phosphorylated forms of the carboxy-terminal domain of RNA-polymerase II. J. Biol. Chem. published online, doi:10.1074/jbc.M803540200 (11 June 2008).

  48. Kapranov, P., Willingham, A.T. & Gingeras, T.R. Genome-wide transcription and the implications for genomic organization. Nat. Rev. Genet. 8, 413–423 (2007).

    Article  CAS  PubMed  Google Scholar 

  49. Van Duyne, G.D., Standaert, R.F., Karplus, P.A., Schreiber, S.L. & Clardy, J. Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin. J. Mol. Biol. 229, 105–124 (1993).

    Article  CAS  PubMed  Google Scholar 

  50. Pei, Y., Hausmann, S., Ho, C.K., Schwer, B. & Shuman, S. The length, phosphorylation state, and primary structure of the RNA polymerase II carboxyl-terminal domain dictate interactions with mRNA capping enzymes. J. Biol. Chem. 276, 28075–28082 (2001).

    Article  CAS  PubMed  Google Scholar 

  51. Wilson, S.M., Datar, K.V., Paddy, M.R., Swedlow, J.R. & Swanson, M.S. Characterization of nuclear polyadenylated RNA-binding proteins in Saccharomyces cerevisiae. J. Cell Biol. 127, 1173–1184 (1994).

    Article  CAS  PubMed  Google Scholar 

  52. Keogh, M.C. & Buratowski, S. Using chromatin immunoprecipitation to map cotranscriptional mRNA processing in Saccharomyces cerevisiae. Methods Mol. Biol. 257, 1–16 (2004).

    CAS  PubMed  Google Scholar 

  53. Reinstein, J. et al. Fluorescence and NMR investigations on the ligand binding properties of adenylate kinases. Biochemistry 29, 7440–7450 (1990).

    Article  CAS  PubMed  Google Scholar 

  54. Efron, B. The Jacknife, the Bootstrap, and other resampling plans. in Society of Industrial and Applied Mathematics CBMS-NSF Monographs Vol. 38 (Cambridge University Press, New York, NY, 1982).

    Google Scholar 

  55. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993).

    Article  CAS  Google Scholar 

  56. Terwilliger, T.C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D Biol. Crystallogr. 55, 849–861 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Terwilliger, T.C. Automated structure solution, density modification and model building. Acta Crystallogr. D Biol. Crystallogr. 58, 1937–1940 (2002).

    Article  PubMed  Google Scholar 

  58. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  PubMed  Google Scholar 

  59. Brunger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

    Article  CAS  PubMed  Google Scholar 

  60. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    Article  CAS  PubMed  Google Scholar 

  61. Thompson, J.D., Higgins, D.G. & Gibson, T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank J. Corden (Johns Hopkins University), D. Libri (Centre National de la Recherche Scientifique) and E. Steinmetz and D. Brow (Univeristy of Wisconsin, Madison) for yeast strains, plasmids and antibodies. We also thank D. Libri, J. Corden, D. Brow, R. Shoeman, Y. Groemping, J. Reinstein, B. Loll and I. Schlichting for helpful discussions, encouragement and support. We are grateful to M. Gebhardt for technical support, I. Vetter for support of the crystallographic software, W. Blankenfeldt for help during data collection, and the scientific staff for support at the beamline X10SA, Paul Scherrer Institute (Villigen, Switzerland). This research was supported by grants to S.B. from the US National Institutes of Health and to A.M. from the German Research Foundation. M.K. is supported by the Charles A. King Trust Postdoctoral Fellowship. L.V. is a recipient of a Special Fellowship from the Leukemia and Lymphoma Society.

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  1. Minkyu Kim and Hannes Mutschler: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Lidia Vasiljeva, Minkyu Kim & Stephen Buratowski

  2. Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, Heidelberg, 69120, Germany

    Hannes Mutschler & Anton Meinhart

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  1. Lidia Vasiljeva
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Contributions

L.V. performed CTD peptide pull-downs, Nrd1–Nab3–Pol II co-precipitations and RNA analysis; M.K. performed the ChIP experiments and constructed several yeast strains; H.M. performed the fluorescence anisotropy experiments and calculated Kd values; A.M. crystallized and solved the structure of the Nrd1 CID. L.V., A.M. and S.B. directed the research and wrote the paper.

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Correspondence to Stephen Buratowski.

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Vasiljeva, L., Kim, M., Mutschler, H. et al. The Nrd1–Nab3–Sen1 termination complex interacts with the Ser5-phosphorylated RNA polymerase II C-terminal domain. Nat Struct Mol Biol 15, 795–804 (2008). https://doi.org/10.1038/nsmb.1468

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