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mRNA interactome capture

Reference Number TO 03-00359

Challenge

RNA-binding proteins (RBP) are RNA binding proteins which have an important function in post-transcriptional regulation of RNA. RBPs control the fate of bound RNA by regulation of RNA metabolism, splicing, transport or decay. Apart from their function in processing mRNA, RBPs also regulate long intervening noncoding RNAs (lincRNAs) by complex interaction networks. LincRNAs are involved in diverse physiological functions and have important functions e.g. in regulation of gene transcription or post-transcriptional mRNA processing. Numerous RBPs meanwhile have been identified to play an important role in human diseases. Considering estimations that the human genome may encode around 800 RBPs, a vast number of their specific functions remain so far unexplored.

mRNA interactome capture workflow
mRNA interactome capture workflow

Commercial Opportunity

To this end various methods have been developed to identify RBPs and their specific RNA binding sites which are all limited to examination of a single RBP. In contrast, mRNA interactome capture allows to capture all protein-RNA interactions occurring in a transcriptome at a given time point for the specific cell. The method combines a crosslinking and immunoprecipitation step (CLIP) with a specific work-up followed by RNA sequencing. Like in the PAR-CLIP approach (photoactivatable-ribonucleoside-enhanced CLIP), crosslinking of RNA with RBPs can be achieved by UV radiation. Cells are provided with photoreactive 4-thiouridine, leading later in the workflow to a typical T-C mutation in the crosslinked binding-site. Key steps in the method are oligo (dT) affinity purification and a specific work-up to reduce unbound RNA leading to a high signal to noise ratio. Thus, the method enables the identification of the global network of protein-RNA interaction in a highly complex mixture such as concentrated cell extracts. This allows e.g. to monitor differential changes in protein-RNA interaction due to internal or external stimuli and disease.

Patent Situation

EP patent application EP2825890pending (priority: March, 2012)

US patent application US2015045237pending (priority: March, 2012)

Further Reading

Methods 2017, doi: 10.1016/j.ymeth.2017.07.006

Methods 2014;65(3);302-9