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RNA Biol. 2014;11(2):146-55. doi: 10.4161/rna.27991. Epub 2014 Feb 7.

Crosslinking-immunoprecipitation (iCLIP) analysis reveals global regulatory roles of hnRNP L.

Author information

1
Institute of Biochemistry; University of Giessen; Giessen, Germany.
2
Kharkevich Institute for Information Transmission Problems; Russian Academy of Sciences; Moscow, Russia; Department of Bioengineering and Bioinformatics; Lomonosov Moscow State University; Moscow, Russia.
3
Institute of Molecular Biology (IMB); Mainz, Germany; Institute of Neurology; University College London; London, United Kingdom.
4
Faculty of Computer and Information Science; University of Ljubljana; Ljubljana, Slovenia.
5
Institute of Neurology; University College London; London, United Kingdom.

Abstract

Heterogeneous nuclear ribonucleoprotein L (hnRNP L) is a multifunctional RNA-binding protein that is involved in many different processes, such as regulation of transcription, translation, and RNA stability. We have previously characterized hnRNP L as a global regulator of alternative splicing, binding to CA-repeat, and CA-rich RNA elements. Interestingly, hnRNP L can both activate and repress splicing of alternative exons, but the precise mechanism of hnRNP L-mediated splicing regulation remained unclear. To analyze activities of hnRNP L on a genome-wide level, we performed individual-nucleotide resolution crosslinking-immunoprecipitation in combination with deep-sequencing (iCLIP-Seq). Sequence analysis of the iCLIP crosslink sites showed significant enrichment of C/A motifs, which perfectly agrees with the in vitro binding consensus obtained earlier by a SELEX approach, indicating that in vivo hnRNP L binding targets are mainly determined by the RNA-binding activity of the protein. Genome-wide mapping of hnRNP L binding revealed that the protein preferably binds to introns and 3' UTR. Additionally, position-dependent splicing regulation by hnRNP L was demonstrated: The protein represses splicing when bound to intronic regions upstream of alternative exons, and in contrast, activates splicing when bound to the downstream intron. These findings shed light on the longstanding question of differential hnRNP L-mediated splicing regulation. Finally, regarding 3' UTR binding, hnRNP L binding preferentially overlaps with predicted microRNA target sites, indicating global competition between hnRNP L and microRNA binding. Translational regulation by hnRNP L was validated for a subset of predicted target 3'UTRs.

KEYWORDS:

CLIP; hnRNP L; microRNA; splicing regulation

PMID:
24526010
PMCID:
PMC3973733
DOI:
10.4161/rna.27991
[Indexed for MEDLINE]
Free PMC Article

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