Detection of RNA-Protein Interactions in Living Cells with SHAPE

Biochemistry. 2015 Nov 24;54(46):6867-75. doi: 10.1021/acs.biochem.5b00977. Epub 2015 Nov 11.

Abstract

SHAPE-MaP is unique among RNA structure probing strategies in that it both measures flexibility at single-nucleotide resolution and quantifies the uncertainties in these measurements. We report a straightforward analytical framework that incorporates these uncertainties to allow detection of RNA structural differences between any two states, and we use it here to detect RNA-protein interactions in healthy mouse trophoblast stem cells. We validate this approach by analysis of three model cytoplasmic and nuclear ribonucleoprotein complexes, in 2 min in-cell probing experiments. In contrast, data produced by alternative in-cell SHAPE probing methods correlate poorly (r = 0.2) with those generated by SHAPE-MaP and do not yield accurate signals for RNA-protein interactions. We then examine RNA-protein and RNA-substrate interactions in the RNase MRP complex and, by comparing in-cell interaction sites with disease-associated mutations, characterize these noncoding mutations in terms of molecular phenotype. Together, these results reveal that SHAPE-MaP can define true interaction sites and infer RNA functions under native cellular conditions with limited preexisting knowledge of the proteins or RNAs involved.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acylation
  • Animals
  • Binding Sites
  • Cells, Cultured
  • Embryonic Stem Cells / metabolism
  • Mice
  • Models, Molecular
  • Mutation
  • Nucleic Acid Conformation
  • Protein Conformation
  • Proteins / chemistry*
  • Proteins / metabolism
  • RNA / chemistry*
  • RNA / genetics
  • RNA / metabolism
  • Ribonucleoproteins / chemistry
  • Ribonucleoproteins / metabolism

Substances

  • Proteins
  • Ribonucleoproteins
  • RNA