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Nat Protoc. 2014 Feb;9(2):263-93. doi: 10.1038/nprot.2014.012. Epub 2014 Jan 9.

Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis.

Author information

1
1] Laboratory of Molecular Neuro-Oncology, The Rockefeller University, New York, New York, USA. [2].
2
1] Laboratory of Molecular Neuro-Oncology, The Rockefeller University, New York, New York, USA. [2] [3].
3
Laboratory of Molecular Neuro-Oncology, The Rockefeller University, New York, New York, USA.

Abstract

The identification of sites where RNA-binding proteins (RNABPs) interact with target RNAs opens the door to understanding the vast complexity of RNA regulation. UV cross-linking and immunoprecipitation (CLIP) is a transformative technology in which RNAs purified from in vivo cross-linked RNA-protein complexes are sequenced to reveal footprints of RNABP:RNA contacts. CLIP combined with high-throughput sequencing (HITS-CLIP) is a generalizable strategy to produce transcriptome-wide maps of RNA binding with higher accuracy and resolution than standard RNA immunoprecipitation (RIP) profiling or purely computational approaches. The application of CLIP to Argonaute proteins has expanded the utility of this approach to mapping binding sites for microRNAs and other small regulatory RNAs. Finally, recent advances in data analysis take advantage of cross-link-induced mutation sites (CIMS) to refine RNA-binding maps to single-nucleotide resolution. Once IP conditions are established, HITS-CLIP takes ∼8 d to prepare RNA for sequencing. Established pipelines for data analysis, including those for CIMS, take 3-4 d.

PMID:
24407355
PMCID:
PMC4156013
DOI:
10.1038/nprot.2014.012
[Indexed for MEDLINE]
Free PMC Article

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