RNA-binding proteins (RBPs) aren’t taking a backseat to DNA binding proteins anymore. RNA analysis has taken the best of chromatin analysis, adapted it, and made it its own. RBPs mediate critical RNA-based processes such as alternative splicing, polyadenylation, sub-cellular localization, translation, and miRNA regulation (Ho and Marsden, 2014). Mutations in these proteins can lead to multiple human diseases and disorders (Lukong et al., 2008).
Technologies to examine RNA-protein interaction have been developed from DNA-protein interaction technologies which share the same principles. The two major approaches are RNA Immunoprecipitation (RIP) and Cross-Linking Immunoprecipitation (CLIP). Both RIP and CLIP are similar to DNA-based ChIP in that they use antibodies to isolate specific nucleic acid-protein interactions.
RNA:Protein Interaction Methods
RIP (RNA Immunoprecipitation) and CLIP (Cross-Linking Immunoprecipitation): 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-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.
RNA:Protein Interaction Additional Reading
This review is a good introduction to RBP biology. The authors cover the domain structure, regulation, and biological functions of RBPs.
- Ho, J.J., and Marsden, P.A. (2014). Competition and collaboration between RNA-binding proteins and microRNAs. Wiley Interdiscip. Rev. RNA 5, 69-86.
- Lukong, K.E., Chang, K.W., Khandjian, E.W., and Richard, S. (2008). RNA-binding proteins in human genetic disease. Trends Genet. 24, 416-425.