Abstract
Cross-linking immunoprecipitation coupled with high-throughput sequencing (CLIP-Seq) has made it possible to identify the targeting sites of RNA-binding proteins in various cell culture systems and tissue types on a genome-wide scale. Here we present a novel model-based approach (MiClip) to identify high-confidence protein-RNA binding sites from CLIP-seq datasets. This approach assigns a probability score for each potential binding site to help prioritize subsequent validation experiments. The MiClip algorithm has been tested in both HITS-CLIP and PAR-CLIP datasets. In the HITS-CLIP dataset, the signal/noise ratios of miRNA seed motif enrichment produced by the MiClip approach are between 17% and 301% higher than those by the ad hoc method for the top 10 most enriched miRNAs. In the PAR-CLIP dataset, the MiClip approach can identify ∼50% more validated binding targets than the original ad hoc method and two recently published methods. To facilitate the application of the algorithm, we have released an R package, MiClip ( http://cran.r-project.org/web/packages/MiClip/index.html ), and a public web-based graphical user interface software (http://galaxy.qbrc.org/tool_runner?tool_id=mi_clip) for customized analysis.
Highlights
RNA-binding proteins (RBPs) participate in RNA translation, splicing and editing events, and play essential roles in mRNA maturation and downstream regulation of cellular events [1]
CLIP-Seq datasets and mapping The AGO HITS-CLIP dataset described in Chi, et al [9] was downloaded from http://AGO.rockefeller.edu
AGO HITS-CLIP dataset We focus the demonstration of the MiClip method on the AGO
Summary
RNA-binding proteins (RBPs) participate in RNA translation, splicing and editing events, and play essential roles in mRNA maturation and downstream regulation of cellular events [1]. Cross-linking immunoprecipitation coupled with high-throughput sequencing (CLIP-Seq) technique has been developed to study genome-wide RNA-protein interactions [5,6,7]. The general procedures of CLIP include covalently linking RNA with RBP, isolating the bound complex, removing the protein and converting RNA to cDNA for sequencing. HITS-CLIP utilizes UV cross-linking of proteins with RNA and introduces mutations in the sequencing data. The mutations are induced on the cDNAs generated in the reverse transcription step from the RNA fragments when the reverse transcription enzyme incorporates an incorrect nucleotide at the site of the cross-linked nucleotide due to attachment of the remaining residues of the covalently bound protein. PAR-CLIP utilizes photoreactive ribonucleoside analogs for incorporation into RNA and some of the analogs that are cross-linked by UV treatment at a later step will result in specific nucleotide substitution events. The analysis of iCLIP experiments requires distinct procedures and is not considered in the MiClip package
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