RNA structure probing to characterize RNA-protein interactions on low abundance pre-mRNA in living cells

In vivo RNA structure analysis has become a powerful tool in molecular biology, largely due to the coupling of an increasingly diverse set of chemical approaches with high-throughput sequencing. This has resulted in a transition from single target to transcriptome-wide approaches. However, these methods require sequencing depths that preclude studying low abundance targets, which are not sufficiently captured in transcriptome-wide approaches. Here we demonstrate that enrichment of low abundance targets before reverse transcription broadens the range of molecules analyzed and results in improved analysis for low abundance transcripts. In addition, this method is compatible with any choice of chemical adduct or read-out approach. We combine this method with inducible expression of an RBP of interest to study an autoregulated event in the pre-mRNA of the splicing factor, muscleblind-like splicing regulator 1 (MBNL1) in a cellular context.

Automated summary

In vivo RNA structure analysis has evolved into a powerful tool through enrichment of low abundance targets for improved analysis of low abundance transcripts, allowing for studying autoregulated events in pre-mRNA in a cellular context.