Advertisement

In Vivo Cross-Linking Followed by PolyA Enrichment to Identify Yeast mRNA Binding Proteins

  • Sarah F. MitchellEmail author
  • Roy Parker
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)

Abstract

mRNA binding proteins regulate gene expression by controlling the processing, localization, decay, and translation of messenger RNAs (mRNAs). To fully understand this process, it is necessary to identify the complete set of mRNA binding proteins. This work describes a method for the systematic identification of yeast mRNA binding proteins. This method applies in vivo UV cross-linking, affinity pull-down of polyA(+) mRNAs, and analysis by mass spectrometry to identify proteins that directly bind to mRNAs.

Key words

mRNA mRNA binding proteins Yeast PolyA Mass spectrometry 

References

  1. 1.
    Dreyfuss G, Choi YD, Adam SA (1984) Characterization of heterogeneous nuclear RNA–protein complexes in vivo with monoclonal antibodies. Mol Cell Biol 4:1104–1114PubMedCentralPubMedGoogle Scholar
  2. 2.
    Batisse J, Batisse C, Budd A et al (2009) Purification of nuclear poly(A)-binding protein Nab2 reveals association with the yeast transcriptome and a messenger ribonucleoprotein core structure. J Biol Chem 284:34911–34917PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Klass DM, Scheibe M, Butter F et al (2013) Quantitative proteomic analysis reveals concurrent RNA–protein interactions and identifies new RNA-binding proteins in Saccharomyces cerevisiae. Genome Res 23:1028–1038PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Tsvetanova NG, Klass DM, Salzman J et al (2010) Proteome-wide search reveals unexpected RNA-binding proteins in Saccharomyces cerevisiae. PloS One 5:e12671PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Scherrer T, Mittal N, Janga SC et al (2010) A screen for RNA-binding proteins in yeast indicates dual functions for many enzymes. PloS One 5:e15499PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Trcek T, Singer RH (2010) The cytoplasmic fate of an mRNP is determined cotranscriptionally: exception or rule? Genes Dev 24:1827–1831PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Riley KJ, Steitz JA (2013) The “Observer Effect” in genome-wide surveys of protein-RNA interactions. Mol Cell 49:601–604PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Castello A, Fischer B, Eichelbaum K et al (2012) Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 149:1393–1406PubMedCrossRefGoogle Scholar
  9. 9.
    Baltz AG, Munschauer M, Schwanhäusser B et al (2012) The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. Mol Cell 46:674–690PubMedCrossRefGoogle Scholar
  10. 10.
    Mitchell SF, Jain S, She M et al (2013) Global analysis of yeast mRNPs. Nat Struct Mol Biol 20:127–133PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Kwon SC, Yi H, Eichelbaum K et al (2013) The RNA-binding protein repertoire of embryonic stem cells. Nat Struct Mol Biol 20:1122–1130PubMedCrossRefGoogle Scholar
  12. 12.
    Teixeira D (2005) Processing bodies require RNA for assembly and contain nontranslating mRNAs. RNA 11:371–382PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Holcik M, Sonenberg N (2005) Translational control in stress and apoptosis. Nat Rev Mol Cell Biol 6:318–327PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryUniversity of Colorado BoulderBoulderUSA
  2. 2.Howard Hughes Medical InstituteUniversity of Colorado BoulderBoulderUSA

Personalised recommendations