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HITS‐CLIP: panoramic views of protein–RNA regulation in living cells

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Abstract The study of gene regulation in cells has recently begun to shift from a period dominated by the study of transcription factor‐DNA interactions to a new focus on RNA regulation. This was sparked by the still‐emerging recognition of the central role for RNA in cellular complexity emanating from the RNA World hypothesis, and has been facilitated by technologic advances, in particular high throughput RNA sequencing and crosslinking methods (RNA‐Seq, CLIP, and HITS‐CLIP). This study will place these advances in context, and, focusing on CLIP, will explain the method, what it can be used for, and how to approach using it. Examples of the successes, limitations, and future of the technique will be discussed. Copyright © 2010 John Wiley & Sons, Ltd. This article is categorized under: RNA Methods > RNA Analyses In Vitro and In Silico RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Methods > RNA Analyses in Cells

Crosslinking immunoprecipitation (CLIP). Tissue (e.g., brain) is UV‐irradiated, covalently crosslinking RNA–protein complexes in situ. After cell lysis and partial RNase digestion to reduce crosslinked RNA to a modal size of ∼50 nt, protein–RNA complexes are purified, protein removed with proteinase K, RNA linkers added and RT‐PCR products sequenced to identify CLIP ‘tags’ that can be mapped onto the transcriptome, delineating native RNA–protein interaction sites. These point to functional binding sites, which can be validated in the tissue used for CLIP, as illustrated here for a Nova splicing target transcript encoding neogenin (Reprinted with permission from Ref 37. Copyright 2003 Science).

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Position‐dependent binding of RNABPs determines the outcome of splicing regulation. The map is a schematic based on HITS‐CLIP studies of Nova, PTB, hnRNP C, and Fox2; see Figure 3 for comparison. The most consistent effects seen (tallest arrows) reveal that binding proximal to or within alternate exons (middle exon) inhibits exon inclusion (blue arrows); binding downstream enhances exon inclusion (red arrows).

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Schematic of Ago HITS‐CLIP. UV‐crosslinking of intact tissues creates covalent bonds between Ago‐miRNA and, separately, Ago in contact with neighboring mRNA sequences (Reprinted with permission from Ref 94). Alignment of the latter produces an Ago‐mRNA footprint; analysis of this restricted sequence space allows prediction and validation of miRNA binding sites (Reprinted with permission from Ref 62. Copyright 2009 Nature).

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Nova regulation of alternative polyadenylation. Nova tags surrounding alternative polyadenylation sites are mapped. CLIP tags from transcripts in which Nova is associated with polyadenylation site skipping or inclusion are shown in top and bottom, respectively (Reprinted with permission from Ref 48. Copyright 2008 Nature).

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A genome‐wide functional Nova interaction map. CLIP tags are plotted around a composite alternate exon, with each unique color representing tags from an individual Nova‐regulated transcript. Tags from transcripts in which Nova inhibits or enhances alternative exon inclusion are represented in cool and warm colors, respectively. Normalization of this data to complexity further refined this map and demonstrated that Nova–RNA interaction sites mapped with HITS‐CLIP experimentally confirm predictions from a previously generated bioinformatic map (Reprinted with permission from Ref 48. Copyright 2008 Nature).

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HITS‐CLIP tags in the NMDAR1 (Grin1) gene. Top panel shows locations of Nova CLIP tag clusters (black bars) across the entire Grin1 transcript. Bottom panels show individual tags, with each color representing a different biologic replicate (individual mouse brain). The bottom right panel, depicting a previously validated Nova‐dependent exon (E24), shows CLIP tags downstream of the alternate exon, which is included in a Nova‐dependent manner (as shown by RT‐PCR of WT vs Nova KO brain). The bottom left panel shows a newly discovered Nova‐regulated exon (E4), in which the position of CLIP tags predicts Nova‐mediated inhibition of exon inclusion according to a previously generated bioinformatic map, which was again validated by RT‐PCR (Reprinted with permission from Ref 48. Copyright 2008 Nature).

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RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes
RNA Methods > RNA Analyses In Vitro and In Silico
RNA Methods > RNA Analyses in Cells
Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs

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