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Bioinformatic tools for analysis of CLIP ribonucleoprotein data

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Investigating the interactions of RNA‐binding proteins (RBPs) with RNAs is a complex task for molecular and computational biologists. The molecular biology techniques and the computational approaches to understand RBP–RNA (or ribonucleoprotein, RNP) interactions have advanced considerably over the past few years and numerous and diverse software tools have been developed to analyze these data. Accordingly, laboratories interested in RNP biology face the challenge of choosing adequately among the available software tools those that best address the biological problem they are studying. Here, we focus on state‐of‐the‐art molecular biology techniques that employ crosslinking and immunoprecipitation (CLIP) of an RBP to study and map RNP interactions. We review the different software tools and databases available to analyze the most widely used CLIP methods, HITS‐CLIP, PAR‐CLIP, and iCLIP. WIREs RNA 2017, 8:e1404. doi: 10.1002/wrna.1404 This article is categorized under: RNA Evolution and Genomics > Computational Analyses of RNA RNA Evolution and Genomics > Ribonomics RNA Methods > RNA Analyses In Vitro and In Silico
Schematic overview of the strategies to identify the sites of RBP‐RNA interactions: HITS‐CLIP, PAR‐CLIP, and iCLIP/eCLIP. All strategies initially involve crosslinking with ultraviolet light (UV) at 254 nm or 365 nm and RNP immunoprecipitation. After RNase digestion and IP, the complexes are digested with protease to release the bound RNA segments, which are used to generate a library for sequencing. PAR‐CLIP analysis includes the pre‐incubation of cells with 4SU to gain further confidence that the binding sites identified are regions of bona fide interaction with RBPs, and iCLIP/eCLIP analysis truncates fragments at the precise site of interaction with the RBP.
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Overview of the general analysis workflow for CLIP‐seq data. (I) The user begins by querying whether an RNP complex of interest has already been reported by searching databases like Starbase, CLIPdb, and doRiNA. (II) If the RNP has not been reported, then the user analyzes or generates a dataset that detects these interactions; the ensuing analysis (gray boxes) begins with preparation of the raw sequencing file, pre‐processing and alignment of the data, and peak calling (identification of the binding site). At that point (white boxes), the binding sites may be further analyzed (comparison, visualization, and identification of motifs, microRNA interaction sites, structural elements, etc). Once the analyses have been completed, they can be made available for other users.
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RNA Evolution and Genomics > Ribonomics
RNA Evolution and Genomics > Computational Analyses of RNA
RNA Methods > RNA Analyses In Vitro and In Silico

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