Characterization of the B Cell Transcriptome Bound by RNA-Binding Proteins with iCLIP

Methods Mol Biol. 2017;1623:159-179. doi: 10.1007/978-1-4939-7095-7_14.

Abstract

Posttranscriptional regulation of gene expression shapes the B cell transcriptome and controls messenger RNA (mRNA) translation into protein. Recent reports have highlighted the importance of RNA binding proteins (RBPs) for mRNA splicing, subcellular location, stability, and translation during B lymphocyte development, activation, and differentiation. Here we describe individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) in primary lymphocytes, a method that maps RNA-protein interactions in a genome-wide scale allowing mechanistic analysis of RBP function. We discuss the latest improvements in iCLIP technology and provide some examples of how integration of the RNA-protein interactome with other high-throughput mRNA sequencing methodologies uncovers the important role of RBP-mediated RNA regulation in key biological cell processes.

Keywords: 3′ Untranslated regions (UTRs); Individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP); Intron; Primary B cells; RNA binding proteins; mRNA maturation; mRNA stability; mRNA translation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • B-Lymphocytes / immunology
  • B-Lymphocytes / metabolism*
  • Binding Sites
  • Cell Separation / methods
  • Computational Biology / methods
  • Gene Expression Profiling* / methods
  • Gene Library
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Immunoprecipitation* / methods
  • Introns
  • Lymphocyte Activation
  • Protein Binding
  • RNA Stability
  • RNA-Binding Proteins / metabolism*
  • Transcriptome*
  • Ultraviolet Rays*

Substances

  • RNA-Binding Proteins