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RNA binding protein mapping

RNA-protein interaction is important in controlling many aspects of gene regulation. In fact, different proteins are required for mRNA processing, such as splicing. What’s more, the properly cellular functions of several non-coding RNAs (ncRNAs) depend upon the formation of RNA-protein complexes.

To identify these interactions, different techniques have been developed. The predominant ones are ‘protein centric’, which require the knowledge of the protein which is analysed. They are based on the purification of proteins followed by the sequencing of RNA molecules associated with them, in order to map the RNA binding proteins (RBPs) across the transcriptome.

RNA immunoprecipitation (RIP)

A first example is the RNA immunoprecipitation (RIP) technique. This method is an antibody-based technique which purifies RNA-protein complexes under physiological conditions. The protein of interest is immunoprecipitated together with its associated RNA, which can then be purified, reverse transcribed in cDNA, amplified with PCR and sequenced.

The advantage of this technique is that  the native complexes present in the cells are preserved. Yet, this kind of technique has some limitations, between them the most concerning is the possibility of formation of non-physiological RNA-protein interactions in solution, in fact the re-association of molecules after cell lysis is possible.

Cross-linking immunoprecipitation (CLIP)

To distinguish between in vivo interactions and interactions that form in solution, RNA-protein complexes are cross-linked in cells, in order to create covalent linkage between physically interacting RNA and proteins. In the CLIP technique this is done using short wavelenght UV light. Then, RNA-protein complexes are purified using stringent wash conditions followed by denaturation of all complexes by heating in sodium dodecyl sulphate (SDS), running the samples on an SDS-polyacrylamide gel electrophoresis (PAGE) gel, and extracting the crosslinked RNA-protein complex, which will run at a size slightly larger than the protein itself, from the gel.

References:

Colleen A McHugh, Pamela Russell and Mitchell Guttman, Methods for comprehensive experimental identification of RNA-protein interactions. Genome Biol. 2014; 15(1): 203.