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

  • Manuel D. Díaz-MuñozEmail author
  • Elisa Monzón-Casanova
  • Martin TurnerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1623)

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.

Key words

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

Notes

Acknowledgments

We thank Prof. Jernej Ule and Prof. Tomaz Curk for their valuable contribution in experimental setup and analysis. The authors were supported by a Biotechnology and Biological Sciences Research Council strategic Lola grant BB/J00152X/1 and program funding BBS/E/B/000C0409.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Laboratory of Lymphocyte Signalling and DevelopmentThe Babraham InstituteCambridgeUK
  2. 2.Department of Immunobiology, Division of Immunology, Infection and Inflammatory DiseaseKing’s College LondonLondonUK
  3. 3.Department of BiochemistryUniversity of CambridgeCambridgeUK

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