Biochemical approach for isolation of polyadenylated RNAs with bound proteins from yeast

STAR Protoc. 2021 Nov 1;2(4):100929. doi: 10.1016/j.xpro.2021.100929. eCollection 2021 Dec 17.

Abstract

In vivo characterization of RNA-protein interactions is the key for understanding RNA regulatory mechanisms. Herein, we describe a protocol for detection of proteins interacting with polyadenylated RNAs in the yeast Saccharomyces cerevisiae. Proteins are crosslinked to nucleic acids in vivo by ultraviolet (UV) irradiation of cells, and poly(A)-containing RNAs with bound proteins are isolated from cell lysates using oligo[dT]25 beads. RBPs can be detected by immunoblot analysis or with mass spectrometry to define the mRNA-binding proteome (mRBPome) and its changes under stress. For complete details on the use and execution of this protocol, please refer to Matia-González et al. (2021, 2015).

Keywords: Gene Expression; Model Organisms; Molecular Biology; Protein Biochemistry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Mass Spectrometry / methods*
  • Protein Interaction Mapping
  • Proteome
  • Proteomics
  • RNA, Fungal* / analysis
  • RNA, Fungal* / chemistry
  • RNA, Fungal* / isolation & purification
  • RNA, Fungal* / metabolism
  • RNA, Messenger* / analysis
  • RNA, Messenger* / chemistry
  • RNA, Messenger* / isolation & purification
  • RNA, Messenger* / metabolism
  • RNA-Binding Proteins* / chemistry
  • RNA-Binding Proteins* / metabolism
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins* / chemistry
  • Saccharomyces cerevisiae Proteins* / metabolism

Substances

  • Proteome
  • RNA, Fungal
  • RNA, Messenger
  • RNA-Binding Proteins
  • Saccharomyces cerevisiae Proteins