Identify RNA-RBP Direct Interactions in Genome-wide Scale

Fast Introduction to HITS-CLIP (or CLIP-seq)

Yun YAN

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HITS-CLIP (or CLIP-seq)

HIThroughput Sequencing of RNAs isolated by CrossLinking ImmunoPrecipitation

Long Definition

boring, huh?!

Well, for Short

CLIP-Seq = Chip-Seq

but RNA version :-P

CLIP-seq = CLIP + Sequencing

Standard CLIP-seq Pipeline

UV-Crosslink >> IP >> Purify >> Sequencing

An RNA code for the FOX2 splicing regulator revealed by mapping RNA-protein interactions in stem cells. NSMB. 2009

Why CLIP?

Identify candidate target RNA

• RIP-Chip, indirect or non-phisiological interactions

• UV-Crosslink and IP, Direct interactions

What Will CLIP-seq Data Offer?

Precise Binding Position

Besides candidate target RNA motif

Robert B. Darnell, etc. CLIP identifies Nova-Regulated RNA networks in the brain. Science. 2003.

Proteins Potentially recuited to machinery

Tag detected + Significant difference between Ctrl/Knock-out:

It is indicated that NOVA is recuited to influence splicing.

Robert B. Darnell, etc. CLIP identifies Nova-Regulated RNA networks in the brain. Science. 2003.

CLIP Update

iCLIP

PAR-CLIP

individual-nucleotide resolution CLIP

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution. J Vis Exp. 2011.

Classic CLIP:

• cDNAs truncate prematurely at the crosslinked nucleotide
• Truncated cDNAs are lost during the standard CLIP library preparation protocol

iCLIP:

More efficient intramolecular cDNA circularization instead of intermolecular RNA ligation to capture truncated cDNAs

Photoactivatable-Ribonucleoside-Enhanced CLIP

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins, J Vis Exp. 2010.

Classic CLIP:

• Low efficiency of UV 254 nm RNA-protein crosslinking
• Difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample

PAR-CLIP:

Rely on 4-thiouridine (4-SU) or 6-thioguanosine (6-SG)

• UV 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs
• 4-SU: T->C transitions
• 6-SG: G->A mutations. The presense of the changes can be assessed during bioinformatic analysis, and make it possible to seperate them from the background of sequences derived from abundant cellular RNAs.

Summary

• HITS-CLIP (or CLIP-seq)
• Exact binding position
• Direct interaction, candiate target RNA
• iCLIP and PAR-CLIP

Check source code at: https://github.com/Puriney/clipseq