RaPID: an aptamer-based mRNA affinity purification technique for the identification of RNA and protein factors present in ribonucleoprotein complexes

Methods Mol Biol. 2011;714:387-406. doi: 10.1007/978-1-61779-005-8_24.

Abstract

RNA metabolism involves regulatory processes, such as transcription, splicing, nuclear export, transport and localization, association with sites of RNA modification, silencing and decay, and necessitates a wide variety of diverse RNA-interacting proteins. These interactions can be direct via RNA-binding proteins (RBPs) or indirect via other proteins and RNAs that form ribonucleoprotein complexes that together control RNA fate. While pull-down methods for the isolation of known RBPs are commonly used, strategies have also been described for the direct isolation of messenger RNAs (mRNAs) and their associated factors. The latter techniques allow for the identification of interacting proteins and RNAs, but most suffer from problems of low sensitivity and high background. Here we describe a simple and highly effective method for RNA purification and identification (RaPID) that allows for the isolation of specific mRNAs of interest from yeast and mammalian cells, and subsequent analysis of the associated proteins and RNAs using mass spectrometry and reverse transcription-PCR, respectively. This method employs the MS2 coat RBP fused to both GFP and streptavidin-binding protein to precipitate MS2 aptamer-tagged RNAs using immobilized streptavidin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aptamers, Nucleotide / metabolism*
  • Capsid Proteins / genetics
  • Capsid Proteins / metabolism
  • Cell Extracts
  • Chemical Fractionation / methods*
  • Culture Techniques
  • Deoxyribonucleases / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • HEK293 Cells
  • Humans
  • RNA, Messenger / genetics
  • RNA, Messenger / isolation & purification*
  • RNA, Messenger / metabolism*
  • Reverse Transcriptase Polymerase Chain Reaction
  • Ribonucleoproteins / isolation & purification*
  • Ribonucleoproteins / metabolism*
  • Saccharomyces cerevisiae / cytology
  • Saccharomyces cerevisiae / growth & development
  • Silver Staining
  • Transfection

Substances

  • Aptamers, Nucleotide
  • Capsid Proteins
  • Cell Extracts
  • RNA, Messenger
  • Ribonucleoproteins
  • Deoxyribonucleases