RNA interactome capture in yeast

Methods. 2017 Apr 15;118-119:82-92. doi: 10.1016/j.ymeth.2016.12.008. Epub 2016 Dec 16.

Abstract

RNA-binding proteins (RBPs) are key players in post-transcriptional regulation of gene expression in eukaryotic cells. To be able to unbiasedly identify RBPs in Saccharomyces cerevisiae, we developed a yeast RNA interactome capture protocol which employs RNA labeling, covalent UV crosslinking of RNA and proteins at 365nm wavelength (photoactivatable-ribonucleoside-enhanced crosslinking, PAR-CL) and finally purification of the protein-bound mRNA. The method can be easily implemented in common workflows and takes about 3days to complete. Next to a comprehensive explanation of the method, we focus on our findings about the choice of crosslinking in yeast and discuss the rationale of individual steps in the protocol.

Keywords: RBP; RNA labeling; UV crosslinking; Yeast; mRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies / chemistry
  • Base Sequence
  • Binding Sites
  • Gene Library
  • High-Throughput Nucleotide Sequencing / methods*
  • Immunoprecipitation / methods*
  • Mass Spectrometry / methods
  • Photochemical Processes
  • Protein Binding
  • RNA, Messenger / chemistry*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA-Binding Proteins / genetics*
  • RNA-Binding Proteins / metabolism
  • Ribonucleases / chemistry
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics*
  • Saccharomyces cerevisiae Proteins / metabolism
  • Sequence Analysis, RNA / methods
  • Staining and Labeling / methods*
  • Sulfur Radioisotopes
  • Thiouracil / analogs & derivatives
  • Thiouracil / chemistry
  • Thiouracil / metabolism
  • Transcriptome
  • Ultraviolet Rays

Substances

  • Antibodies
  • RNA, Messenger
  • RNA-Binding Proteins
  • Saccharomyces cerevisiae Proteins
  • Sulfur Radioisotopes
  • 4-thiouracil
  • Thiouracil
  • Ribonucleases