Though it sounds like the catch phrase of a bad 80’s action hero, “RIP and CLIP” are actually the two fundamental approaches for analyzing RNA-protein interactions. RIP is the older technique from which CLIP was derived. RIP relies on the principle that if an RNA and protein are interacting in the cell, the protein likely has some role in regulating the RNA. RIP involves immunoprecipitation (IP) of an RNA-binding protein (RBP) of interest using an antibody. Any RNA bound to the RBP will be isolated in this IP provided that the isolation is done non-stringently. This low stringency results in low specificity leading to the development of CLIP.
CLIP is very similar to RIP, expect it adds a crosslinking step. Unlike DNA-protein crosslinking which is done with formaldehyde, CLIP uses ultraviolet (UV) light. Unlike formaldehyde, UV crosslinking is irreversible. UV crosslinks are also more specific and only link proteins to RNAs that are in very close proximity. Further, UV crosslinks do not form between two proteins (Brimacombe et al., 1988). For these reasons, UV has become the standard in RNA research. The RNA-RBP complexes are then immunoprecipitated. Due to the irreversibility of the crosslinks, the next step is digestion with a proteinase.
In both RIP and CLIP, the isolated RNA is reverse transcribed to cDNA then analyzed with either microarray (RIP-chip) or sequencing (RIP-seq, HITS-CLIP aka CLIP-seq). More specialized techniques have been developed from these: PAR-CLIP improves crosslinking with photoreactive RNA nucleotides, iCLIP uses reverse transcriptase stalling to map individual nucleotide-RBP interactions, and finally miCLIP modifies an RNA methylase to map its binding sites.
RIP and CLIP Methods
RIP-chip & RIP-seq: RIP can be coupled to microarray (RIP-chip) or sequencing (RIP-seq) to identify RNAs that are bound by an RBP of interest. Both rely on the specificity of RBP-RNA interaction for immunoprecipitation.
HITS-CLIP (or CLIP-seq): Stands for high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation, which utilizes in vivo UV crosslinking technologies and next-gen sequencing.
PAR-CLIP: Photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation attempts to solve some of the problems of HITS-CLIP, namely, efficiency of crosslinking and resolution of RBP binding sites.
iCLIP: Individual-nucleotide resolution CLIP is a refinement of CLIP that allows single-nucleotide resolution of RBP binding sites.
miCLIP: Methylation individual-nucleotide-resolution crosslinking and immunoprecipitation is a specialized version of iCLIP designed to determine which RNA nucleotides are methylated by the RNA methyltransferase Nsun2.
RIP and CLIP Additional Reading
This review compares CLIP, HITS-CLIP, iCLIP, and PAR-CLIP. It also lists different studies using these techniques and the RBPs they studied. It also has a good outlook section discussing some of the future challenges in the field.
This paper is a critical examination of RIP and CLIP technologies. It focuses on The Observer Effect, which refers to minor perturbations of the experiment by the observer effecting the outcome.
Reference List
- Brimacombe, R., Stiege, W., Kyriatsoulis, A., and Maly, P. (1988). Intra-RNA and RNA-protein cross-linking techniques in Escherichia coli ribosomes. Methods Enzymol. 164, 287-309.