A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins

@article{Kishore2011AQA,
  title={A quantitative analysis of CLIP methods for identifying binding sites of RNA-binding proteins},
  author={Shivendra Kishore and Lukasz Jaskiewicz and Lukas Burger and Jean Hausser and Mohsen Khorshid and Mihaela Zavolan},
  journal={Nature Methods},
  year={2011},
  volume={8},
  pages={559-564},
  url={https://api.semanticscholar.org/CorpusID:5313093}
}
The results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect their binding sites sufficiently under the denaturing conditions used during the CLIP procedure, and show that extensive digestion with sequence-specific RNases strongly biases the recovered binding sites.
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38 References

CLIPZ: a database and analysis environment for experimentally determined binding sites of RNA-binding proteins

The CLIPZ database and analysis environment, available at http://www.clipz.ch, aims to provide an open access repository of information for post-transcriptional regulatory elements, and can be visualized at the level of the genome and of individual transcripts.

CLIP: a method for identifying protein-RNA interaction sites in living cells.

HITS-CLIP yields genome-wide insights into brain alternative RNA processing

A genome-wide means of mapping protein–RNA binding sites in vivo, by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP), which revealed a large number of Nova–RNA interactions in 3′ untranslated regions, leading to the discovery that Nova regulates alternative polyadenylation in the brain.

Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs

It is demonstrated that the snoRNP proteins directly contact the pre-rRNA substrate, suggesting roles in snoRNA recruitment and the techniques reported here should be widely applicable to analyses of RNA–protein interactions.

Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP

Rapid and systematic analysis of the RNA recognition specificities of RNA-binding proteins

Using in vitro and in vivo binding data, it is demonstrated that preferences for individual 7-mers identified by RNAcompete are a more accurate representation of binding activity than are conventional motif models.

Ago HITS-CLIP decodes miRNA-mRNA interaction maps

High-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation provides a general platform for exploring the specificity and range of miRNA action in vivo, and identifies precise sequences for targeting clinically relevant miRNA–mRNA interactions.

CLIP: construction of cDNA libraries for high-throughput sequencing from RNAs cross-linked to proteins in vivo.

Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans

A striking enrichment of Argonaute binding sites in genes important for miRNA function is revealed, suggesting an autoregulatory role that may confer robustness to the miRNA pathway.

iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution

Individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) data show that hnRNP C recognizes uridine tracts with a defined long-range spacing consistent with hmRNP particle organization, and integration of transcriptome-wide iCLIP data and alternative splicing profiles into an 'RNA map' indicates how the positioning of hn RNP particles determines their effect on the inclusion of alternative exons.