Identification of RNA-Binding Proteins Associated to RNA Structural Elements

Methods Mol Biol. 2021;2323:109-119. doi: 10.1007/978-1-0716-1499-0_9.

Abstract

RNA motifs guide the interaction with specific proteins leading to the assembly of ribonucleoprotein complexes that perform key functions in cellular processes. Internal ribosome entry site (IRES) elements are organized in structural domains that determine internal initiation of translation. In this chapter we describe a pull-down assay using streptavidin-aptamer tagged RNAs that combines RNA structure-dependent protein isolation with proteomic analysis to identify novel interactors recognizing RNA structural domains. This approach takes advantage of tRNA-scaffold guided expression, allowing the identification of factors belonging to networks involved in RNA and protein metabolism.

Keywords: IRES elements; Pull-down; RNA structure; RNA-binding proteins; Streptavidin aptamer; Translation control; tRNA scaffold.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aptamers, Nucleotide
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Internal Ribosome Entry Sites
  • Mass Spectrometry
  • Nucleotide Motifs* / genetics
  • Protein Biosynthesis
  • Proteomics / methods
  • RNA / isolation & purification
  • RNA / metabolism
  • RNA, Transfer / biosynthesis
  • RNA, Transfer / chemistry
  • RNA-Binding Proteins / isolation & purification*
  • RNA-Binding Proteins / metabolism
  • Streptavidin
  • Substrate Specificity

Substances

  • Aptamers, Nucleotide
  • Internal Ribosome Entry Sites
  • RNA, recombinant
  • RNA-Binding Proteins
  • RNA
  • Streptavidin
  • RNA, Transfer