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

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

Author information

1
Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, CB22 3AT, UK. manuel.diaz-munoz@babraham.ac.uk.
2
Department of Immunobiology, Division of Immunology, Infection and Inflammatory Disease, King's College London, SE1 9RT, London, UK. manuel.diaz-munoz@babraham.ac.uk.
3
Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, CB22 3AT, UK.
4
Department of Biochemistry, University of Cambridge, Cambridge, UK.
5
Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, CB22 3AT, UK. martin.turner@babraham.ac.uk.

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

PMID:
28589356
DOI:
10.1007/978-1-4939-7095-7_14
[Indexed for MEDLINE]

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