tRIP‐seq reveals repression of premature polyadenylation by co‐transcriptional FUS‐U1 snRNP assembly

RNA processing occurs co‐transcriptionally through the dynamic recruitment of RNA processing factors to RNA polymerase II (RNAPII). However, transcriptome‐wide identification of protein–RNA interactions specifically assembled on transcribing RNAPII is challenging. Here, we develop the targeted RNA immunoprecipitation sequencing (tRIP‐seq) method that detects protein–RNA interaction sites in thousands of cells. The high sensitivity of tRIP‐seq enables identification of protein–RNA interactions at functional subcellular levels. Application of tRIP‐seq to the FUS‐RNA complex in the RNAPII machinery reveals that FUS binds upstream of alternative polyadenylation (APA) sites of nascent RNA bound to RNAPII, which retards RNAPII and suppresses the recognition of the polyadenylation signal by CPSF. Further tRIP‐seq analyses demonstrate that the repression of APA is achieved by a complex composed of FUS and U1 snRNP on RNAPII, but not by either one alone. Moreover, our analysis reveals that FUS mutations in familial amyotrophic lateral sclerosis (ALS) that impair the FUS‐U1 snRNP interaction aberrantly activate the APA sites. tRIP‐seq provides new insights into the regulatory mechanism of co‐transcriptional RNA processing by RNA processing factors.