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

Abstract

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 cotranscriptional RNA processing by RNA processing factors.

Rapidly Growing Protein-Centric Technologies to Extensively Identify Protein–RNA Interactions: Application to the Analysis of Co-Transcriptional RNA Processing

“…We recently reported the target RNA immunoprecipitation (tRIP) method [ 56 ], in which UV-crosslinked protein–RNA complexes are treated with the Terminator 5′-phosphate-dependent exonuclease (TEX) instead of purification with SDS-PAGE and membrane transfer in CLIP ( Table 1 ). As TEX-dependent digestion of RNA is stopped at a protein-tethered nucleotide, the TEX treatment retains protein-crosslinked RNA but eliminates linkers and non-specific RNA.…”

“…The high sensitivity of tRIP-seq enabled the identification of protein–RNA interaction sites assembled on RNAP II by serial IP of RNAP II–RNA–protein complexes ( Figure 3 ) [ 56 ]. Following UV-crosslinking, the RNAP II machinery, including associated RNA and proteins, is immunoprecipitated from cell lysates using an antibody specific to RNAP II.…”

“…Streamlining several steps in the CLIP protocol (Table 1), including the use of infrared-dye-conjugated RNA adapter for the visualization of the fragmented RNA, and the use of thermostable group II intron reverse transcriptase (TGIRT) for cDNA synthesis, has allowed the productive sequencing of cDNA libraries from 20,000 cells. eCLIP technique also involves the optimization of reaction conditions in RNA and cDNA handling (Table 1), which decreases the requisite PCR-amplification by ≈1000-fold in the cDNA library generation as compared with iCLIP [55], although most eCLIPs were performed using tens of millions of cells [41] We recently reported the target RNA immunoprecipitation (tRIP) method [56], in which UV-crosslinked protein-RNA complexes are treated with the Terminator 5 -phosphatedependent exonuclease (TEX) instead of purification with SDS-PAGE and membrane transfer in CLIP (Table 1). As TEX-dependent digestion of RNA is stopped at a protein-tethered nucleotide, the TEX treatment retains protein-crosslinked RNA but eliminates linkers and non-specific RNA.…”

Noncoding RNAs Set the Stage for RNA Polymerase II Transcription

“…Inhibition of these gene-internal PASs is termed telescripting and requires the U1 snRNP. U1 snRNP bound to 5'SSs and related motifs throughout nascent RNAs prevents the activation of cryptic PASs even several hundred nucleotides away [114][115][116] (Figure 5). Consequently, loss of U1 snRNA induces global shortening of mRNAs, with most prematurely-terminated in the first intron.…”

Shortening of 3’ UTRs in most cell types composing tumor tissues implicates alternative polyadenylation in protein metabolism

“…Various RNA-binding proteins (RBPs) are also involved in specific cellular systems (A. J. Lee et al, 2021;Martin et al, 2012;Masuda et al, 2020;So et al, 2019).…”