RNA-Binding Proteins and Their Targets in Trypanosoma brucei: Single Nucleotide Resolution Using iCLIP and iCLAP

Methods Mol Biol. 2020:2116:303-323. doi: 10.1007/978-1-0716-0294-2_19.

Abstract

RNA-binding proteins (RBPs) are critical to posttranscriptional gene regulation. Therefore, characterization of the RNA molecules bound by RBPs in vivo represent a key step in elucidating their function. The recently developed iCLIP technique allows single nucleotide resolution of the RNA binding footprints of RBPs. We present the iCLIP technique modified for its application to Trypanosoma brucei and most likely other kinetoplastid flagellates. By using the immuno- or affinity purification approach, it was successfully applied to the analysis of several RBPs. Furthermore, we also provide a detailed description of the iCLIP/iCLAP protocol that shall be particularly suitable for the studies of trypanosome RBPs.

Keywords: Posttranscriptional gene regulation; RNA-binding proteins (RBPs); iCLAP; iCLIP.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites / genetics
  • Immunoprecipitation / methods*
  • Nucleotides / genetics
  • Nucleotides / metabolism
  • Parasitology / methods
  • Protein Binding / genetics
  • Protein Binding / radiation effects
  • Protozoan Proteins / analysis*
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism
  • RNA / genetics
  • RNA / metabolism
  • RNA, Protozoan / genetics
  • RNA, Protozoan / metabolism
  • RNA-Binding Proteins / analysis*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • Single Molecule Imaging / methods
  • Trypanosoma brucei brucei / genetics*
  • Ultraviolet Rays

Substances

  • Nucleotides
  • Protozoan Proteins
  • RNA, Protozoan
  • RNA-Binding Proteins
  • RNA