The Future of Cross-Linking and Immunoprecipitation (CLIP)

Abstract

To understand the assembly and functional outcomes of protein-RNA regulation, it is crucial to precisely identify the positions of such interactions. Crosslinking and immunoprecipitation (CLIP) serves this purpose by exploiting covalent protein-RNA crosslinking and RNA fragmentation, along with a series of stringent purification and quality control steps to prepare cDNA libraries for sequencing. Here we describe the core steps of CLIP, its primary variations and the approaches to data analysis. We present the application of CLIP to studies of specific cell types in genetically engineered mice, and discuss the mechanistic and physiologic insights that have already been gained from studies employing CLIP. We conclude by discussing the future opportunities for CLIP, including studies of human postmortem tissues from disease patients and controls, RNA epigenetic modifications, and RNA structure. These and other applications of CLIP will continue to unravel fundamental gene regulatory mechanisms, while providing important biologic and clinically relevant insights.

Occurrence and Functions of m6A and Other Covalent Modifications in Plant mRNA

“…(2) Modified nucleotides are specifically derivatized using unique reactivity with an appropriate compound, and the presence of the derived nucleotide is read either as a mutation signature or as a stop upon reverse transcription (Squires et al, 2012;Carlile et al, 2014;Lovejoy et al, 2014;Schwartz et al, 2014a;Li et al, 2015;David et al, 2017;Enroth et al, 2019;Sun et al, 2019;Zhang et al, 2019b). The cross-linking immunoprecipitation (CLIP) variant miCLIP constitutes an important special case in this category: a modification-specific antibody is UV cross-linked to purified RNA, allowing mutation-dependent mapping of cross-link sites (Ke et al, 2015;Linder et al, 2015) as in other CLIP techniques (Ule et al, 2018). (3) Differential RNA cleavage at modified versus unmodified nucleotides is used to identify modified sites based on the percentage of sequence read-throughs and stops at each nucleotide (Birkedal et al, 2015;Garcia-Campos et al, 2019;Zhang et al, 2019d).…”

FMRP binding to a ranked subset of long genes is revealed by coupled CLIP and TRAP in specific neuronal cell types

“…Moreover, in vivo binding sites are revealed which often suggest function. Most importantly, CLIP is a transcriptome-wide, unbiased approach [47][48][49][50] .…”

“…UTR (and intronic sequences surrounding the last exon)while it may not be as critical for a housekeeping protein like a rpL22. Breeding of the Fmr1 cTAG and RiboTag mice in parallel with the same Cre lines allowed interrogation of both FMRP:RNA interactions and TRAP-Seq quantitation of ribosome-associated mRNAs in the same cell populations at the same ages.We considered many ways to quantify the abundance of individual mRNAs in the same cell types expressing tagged FMRP, including TRAP-Seq85,86 , manual sorting by fluorescence 113 , laser capture microdissection followed by scRNA-Seq 114 , polyA-binding protein (PABP) CLIP48,110,111,115 , immuno-panning, FACS sorting of GFP-labelled nuclei116 , and scRNA-Seq 72 , considering various issues with each of the techniques[117][118][119][120] . Because cell transcriptomes can change following tissue dissociation, and dissociation of neurons can lead to capture of only part of the transcriptome (losing neuronal processes, or capturing only nuclear RNA), and scRNA-Seq can be limited in its ability to capture rare transcripts and preserve relative abundance among mRNAs, we preferred a method that would preserve in vivo interactions, similar to CLIP, and in a cell population as close as possible to that used for FMRP cTAG CLIP.…”

The kinetic landscape of an RNA binding protein in cells

“…Yet, association and dissociation kinetics of RBPs at individual cellular binding sites have not been experimentally accessible. In cells, steady-state patterns of RNA-protein interactions have been measured [3][4][5] . RBP binding and dissociation kinetics have only been determined in vitro 2,6 .…”