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.

  • Resource
  • Published:

Global analysis of yeast mRNPs

Nature Structural & Molecular Biology volume 20, pages 127–133 (2013)Cite this article

Subjects

Abstract

Proteins regulate gene expression by controlling mRNA biogenesis, localization, translation and decay. Identifying the composition, diversity and function of mRNA–protein complexes (mRNPs) is essential to understanding these processes. In a global survey of Saccharomyces cerevisiae mRNA-binding proteins, we identified 120 proteins that cross-link to mRNA, including 66 new mRNA-binding proteins. These include kinases, RNA-modification enzymes, metabolic enzymes and tRNA- and rRNA-metabolism factors. These proteins show dynamic subcellular localization during stress, including assembly into stress granules and processing bodies (P bodies). Cross-linking and immunoprecipitation (CLIP) analyses of the P-body components Pat1, Lsm1, Dhh1 and Sbp1 identified sites of interaction on specific mRNAs, revealing positional binding preferences and co-assembly preferences. When taken together, this work defines the major yeast mRNP proteins, reveals widespread changes in their subcellular location during stress and begins to define assembly rules for P-body mRNPs.

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: Description of the in vivo RBP capture procedure and identified mRNA-binding proteins.
Figure 2: Fluorescence microscopy images to show localization of mRNA-binding proteins fused to GFP under log-phase growth under normal (+Glu) and glucose-deprivation conditions (−Glu).
Figure 3: Categorization of new granule components.
Figure 4: P-body proteins bind to a shared group of mRNAs with positional specificity.
Figure 5: Interactions between P-body proteins influence mRNA binding.

Similar content being viewed by others

References

  1. Schwanhäusser, B. et al. Global quantification of mammalian gene expression control. Nature 473, 337–342 (2011).

    Article  Google Scholar 

  2. Tsvetanova, N.G., Klass, D.M., Salzman, J. & Brown, P.O. Proteome-wide search reveals unexpected RNA-binding proteins in Saccharomyces cerevisiae. PLoS ONE 5, e12671 (2010).

    Article  Google Scholar 

  3. Matunis, M.J., Matunis, E.L. & Dreyfuss, G. PUB1: a major yeast poly(A)+ RNA-binding protein. Mol. Cell. Biol. 13, 6114–6123 (1993).

    Article  CAS  Google Scholar 

  4. Castello, A. et al. Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 149, 1393–1406 (2012).

    Article  CAS  Google Scholar 

  5. Baltz, A.G. et al. The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. Mol. Cell 46, 674–690 (2012).

    Article  CAS  Google Scholar 

  6. Teixeira, D., Sheth, U., Valencia-Sanchez, M.A., Brengues, M. & Parker, R. Processing bodies require RNA for assembly and contain nontranslating mRNAs. RNA 11, 371–382 (2005).

    Article  CAS  Google Scholar 

  7. Holcik, M. & Sonenberg, N. Translational control in stress and apoptosis. Nat. Rev. Mol. Cell Biol. 6, 318–327 (2005).

    Article  CAS  Google Scholar 

  8. Dewey, C.M. et al. TDP-43 aggregation in neurodegeneration: Are stress granules the key? Brain Res. 10.1016/j.brainres.2012.02.032 (2012).

  9. Nogueira, T., de Smit, M., Graffe, M. & Springer, M. The relationship between translational control and mRNA degradation for the Escherichia coli threonyl-tRNA synthetase gene. J. Mol. Biol. 310, 709–722 (2001).

    Article  CAS  Google Scholar 

  10. Frugier, M. & Giegé, R. Yeast aspartyl-tRNA synthetase binds specifically its own mRNA. J. Mol. Biol. 331, 375–383 (2003).

    Article  CAS  Google Scholar 

  11. Sampath, P. et al. Noncanonical function of glutamyl-prolyl-tRNA synthetase: gene-specific silencing of translation. Cell 119, 195–208 (2004).

    Article  CAS  Google Scholar 

  12. Hogan, D.J., Riordan, D.P., Gerber, A.P., Herschlag, D. & Brown, P.O. Diverse RNA-binding proteins interact with functionally related sets of RNAs, suggesting an extensive regulatory system. PLoS Biol. 6, e255 (2008).

    Article  Google Scholar 

  13. Du, T.-G. Nuclear transit of the RNA-binding protein She2 is required for translational control of localized ASH1 mRNA. EMBO Rep. 9, 781–787 (2008).

    Article  CAS  Google Scholar 

  14. Decker, C.J., Teixeira, D. & Parker, R. Edc3p and a glutamine/asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae. J. Cell Biol. 179, 437–449 (2007).

    Article  CAS  Google Scholar 

  15. Buchan, J.R. & Parker, R. Eukaryotic stress granules: the ins and outs of translation. Mol. Cell 36, 932–941 (2009).

    Article  CAS  Google Scholar 

  16. Buchan, J.R., Yoon, J.-H. & Parker, R. Stress-specific composition, assembly and kinetics of stress granules in Saccharomyces cerevisiae. J. Cell Sci. 124, 228–239 (2011).

    Article  CAS  Google Scholar 

  17. Hoyle, N.P., Castelli, L.M., Campbell, S.G., Holmes, L.E.A. & Ashe, M.P. Stress-dependent relocalization of translationally primed mRNPs to cytoplasmic granules that are kinetically and spatially distinct from P-bodies. J. Cell Biol. 179, 65–74 (2007).

    Article  CAS  Google Scholar 

  18. Grousl, T. et al. Robust heat shock induces eIF2alpha-phosphorylation-independent assembly of stress granules containing eIF3 and 40S ribosomal subunits in budding yeast, Saccharomyces cerevisiae. J. Cell Sci. 122, 2078–2088 (2009).

    Article  CAS  Google Scholar 

  19. Sheth, U. & Parker, R. Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 300, 805–808 (2003).

    Article  CAS  Google Scholar 

  20. Nissan, T., Rajyaguru, P., She, M., Song, H. & Parker, R. Decapping activators in Saccharomyces cerevisiae act by multiple mechanisms. Mol. Cell 39, 773–783 (2010).

    Article  CAS  Google Scholar 

  21. Segal, S.P., Dunckley, T. & Parker, R. Sbp1p affects translational repression and decapping in Saccharomyces cerevisiae. Mol. Cell. Biol. 26, 5120–5130 (2006).

    Article  CAS  Google Scholar 

  22. Chowdhury, A., Mukhopadhyay, J. & Tharun, S. The decapping activator Lsm1p-7p-Pat1p complex has the intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs. RNA 13, 998–1016 (2007).

    Article  CAS  Google Scholar 

  23. He, W. & Parker, R. The yeast cytoplasmic LsmI/Pat1p complex protects mRNA 3′ termini from partial degradation. Genetics 158, 1445–1455 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Bailey, T.L. DREME: motif discovery in transcription factor ChIP-seq data. Bioinformatics 27, 1653–1659 (2011).

    Article  CAS  Google Scholar 

  25. Rajyaguru, P., She, M. & Parker, R. Scd6 targets eIF4G to repress translation: RGG motif proteins as a class of eIF4G-binding proteins. Mol. Cell 45, 244–254 (2012).

    Article  CAS  Google Scholar 

  26. Hoell, J.I. et al. RNA targets of wild-type and mutant FET family proteins. Nat. Struct. Mol. Biol. 10.1038/nsmb.2163 (2011).

  27. Coller, J.M., Tucker, M., Sheth, U., Valencia-Sanchez, M.A. & Parker, R. The DEAD box helicase, Dhh1p, functions in mRNA decapping and interacts with both the decapping and deadenylase complexes. RNA 7, 1717–1727 (2001).

    Article  CAS  Google Scholar 

  28. Ghaemmaghami, S. et al. Global analysis of protein expression in yeast. Nature 425, 737–741 (2003).

    Article  CAS  Google Scholar 

  29. Kraft, C., Deplazes, A., Sohrmann, M. & Peter, M. Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease. Nat. Cell Biol. 10, 602–610 (2008).

    Article  CAS  Google Scholar 

  30. Yoon, J.-H., Choi, E.-J. & Parker, R. Dcp2 phosphorylation by Ste20 modulates stress granule assembly and mRNA decay in Saccharomyces cerevisiae. J. Cell Biol. 189, 813–827 (2010).

    Article  CAS  Google Scholar 

  31. Tharun, S. & Parker, R. Targeting an mRNA for decapping: displacement of translation factors and association of the Lsm1p-7p complex on deadenylated yeast mRNAs. Mol. Cell 8, 1075–1083 (2001).

    Article  CAS  Google Scholar 

  32. Hilgers, V., Teixeira, D. & Parker, R. Translation-independent inhibition of mRNA deadenylation during stress in Saccharomyces cerevisiae. RNA 12, 1835–1845 (2006).

    Article  CAS  Google Scholar 

  33. Coller, J. & Parker, R. Eukaryotic mRNA decapping. Annu. Rev. Biochem. 73, 861–890 (2004).

    Article  CAS  Google Scholar 

  34. Swisher, K.D. & Parker, R. Localization to, and effects of Pbp1, Pbp4, Lsm12, Dhh1, and Pab1 on stress granules in Saccharomyces cerevisiae. PLoS ONE 5, e10006 (2010).

    Article  Google Scholar 

  35. Teixeira, D. & Parker, R. Analysis of P-body assembly in Saccharomyces cerevisiae. Mol. Biol. Cell 18, 2274–2287 (2007).

    Article  CAS  Google Scholar 

  36. Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858 (1996).

    Article  CAS  Google Scholar 

  37. Andon, N.L. et al. Proteomic characterization of wheat amyloplasts using identification of proteins by tandem mass spectrometry. Proteomics 2, 1156–1168 (2002).

    Article  CAS  Google Scholar 

  38. Qian, W.-J. et al. Probability-based evaluation of peptide and protein identifications from tandem mass spectrometry and SEQUEST analysis: the human proteome. J. Proteome Res. 4, 53–62 (2005).

    Article  CAS  Google Scholar 

  39. Buchan, J.R., Muhlrad, D. & Parker, R. P bodies promote stress granule assembly in Saccharomyces cerevisiae. J. Cell Biol. 183, 441–455 (2008).

    Article  CAS  Google Scholar 

  40. Ule, J., Jensen, K., Mele, A. & Darnell, R.B. CLIP: a method for identifying protein-RNA interaction sites in living cells. Methods 37, 376–386 (2005).

    Article  CAS  Google Scholar 

  41. Wang, Z., Tollervey, J., Briese, M., Turner, D. & Ule, J. CLIP: construction of cDNA libraries for high-throughput sequencing from RNAs cross-linked to proteins in vivo. Methods 48, 287–293 (2009).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  43. Bailey, T.L. DREME: motif discovery in transcription factor ChIP-seq data. Bioinformatics 27, 1653–1659 (2011).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

MS and proteomics data were acquired by the Arizona Proteomics Consortium supported by the US National Institute of Environmental Health Science grant ES06694 (the S.W.E.H.S.C.), the US National Institutes of Health, US National Cancer Institute grant CA023074 (the A.Z.C.C.) and the BIO5 Institute of the University of Arizona. The Thermo Fisher LTQ Orbitrap Velos mass spectrometer was provided by the US National Institutes of Health, US National Center for Research Resources grant 1S10 RR028868-01 (G.T.). We thank J.R. Buchan for assistance with microscopy and C. Decker and other members of the Parker lab for helpful discussions. This work was supported by funding from the US National Institutes of Health grant 7R37 GM045443 (R.P.), a Howard Hughes Medical Institute grant (R.P.) and Leukemia and Lymphoma Society fellowship 5687-13 (S.F.M.).

Author information

Author notes

  1. Sarah F Mitchell and Saumya Jain: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Colorado Boulder, Boulder, Colorado, USA

    Sarah F Mitchell, Saumya Jain & Roy Parker

  2. Howard Hughes Medical Institute, University of Colorado Boulder, Boulder, Colorado, USA

    Sarah F Mitchell, Meipei She & Roy Parker

  3. Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona, USA

    Saumya Jain & Meipei She

Authors
  1. Sarah F Mitchell
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Saumya Jain
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Meipei She
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Roy Parker
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.P., S.F.M., S.J. and M.S. designed the project. S.F.M. performed the in vivo RBP capture experiments and CLIP analysis, S.J. did the microscopy and CLIP analysis, and M.S. performed the CLIP experiments and analysis. R.P., S.F.M. and S.J. wrote the paper.

Corresponding author

Correspondence to Roy Parker.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Table 2, Supplementary note (PDF 378 kb)

Supplementary Table 1

A list of all 120 proteins identified as enriched by the in vivo capture of RBPs. (XLSX 48 kb)

Supplementary Table 3

A list of mRNAs (Tier1 and Tier2) bound by Dhh1, Lsm1, Pat1 and Sbp1. (XLSX 127 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mitchell, S., Jain, S., She, M. et al. Global analysis of yeast mRNPs. Nat Struct Mol Biol 20, 127–133 (2013). https://doi.org/10.1038/nsmb.2468

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.2468

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