Quantitative identification of proteins that influence miRNA biogenesis by RNA pull-down-SILAC mass spectrometry (RP-SMS)

Methods. 2019 Jan 1:152:12-17. doi: 10.1016/j.ymeth.2018.06.006. Epub 2018 Jun 8.

Abstract

RNA-binding proteins mediate and control gene expression. As some examples, they regulate pre-mRNA synthesis and processing; mRNA localisation, translation and decay; and microRNA (miRNA) biogenesis and function. Here, we present a detailed protocol for RNA pull-down coupled to stable isotope labelling by amino acids in cell culture (SILAC) mass spectrometry (RP-SMS) that enables quantitative, fast and specific detection of RNA-binding proteins that regulate miRNA biogenesis. In general, this method allows for the identification of RNA-protein complexes formed using in vitro or chemically synthesized RNAs and protein extracts derived from cultured cells.

Keywords: Mass spectrometry; RNA; RNA pull-down; RNA-binding protein; miRNA; miRNA biogenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Culture Techniques
  • HeLa Cells
  • Humans
  • Isotope Labeling / methods*
  • Mass Spectrometry / methods*
  • MicroRNAs / biosynthesis*
  • MicroRNAs / chemistry
  • MicroRNAs / metabolism
  • RNA-Binding Proteins / chemistry*
  • RNA-Binding Proteins / isolation & purification

Substances

  • MicroRNAs
  • RNA-Binding Proteins