Sample preparation for mass spectrometry-based identification of RNA-binding regions

Robert Warneford Thomson, Chongsheng He, Simone Sidoli, Benjamin A. Garcia, Roberto Bonasio

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods. Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNAbinding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

Original languageEnglish (US)
Article numbere56004
JournalJournal of Visualized Experiments
Volume2017
Issue number127
DOIs
StatePublished - Sep 28 2017
Externally publishedYes

Keywords

  • Biochemistry
  • Issue 127
  • Mass spectrometry
  • Noncoding RNAs
  • Protein-RNA interactions
  • RNA-binding domain
  • RNA-binding region
  • UV crosslinking

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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