Capture RIC-seq reveals positional rules of PTBP1-associated RNA loops in splicing regulation [CRIC-seq]

RNA-binding proteins (RBPs) bind at different positions of pre-mRNA molecules to promote or reduce the usage of a particular exon. Seeking to understand the working principle of these positional effects, we develop a capture RIC-seq (CRIC-seq) method to enrich specific RBP-associated in situ proximal RNA-RNA fragments for deep sequencing. We determine hnRNPA1-, SRSF1-, and PTBP1-associated proximal RNA-RNA contacts and regulatory mechanisms in HeLa cells. Unexpectedly, the 3D RNA map analysis shows that PTBP1-associated loops in individual introns preferentially promote cassette exon splicing by accelerating asymmetric intron removal, whereas the loops spanning across cassette exon primarily repress splicing. These “positional rules” can faithfully predict PTBP1-regulated splicing outcomes. We further demonstrate that cancer-related splicing quantitative trait loci can disrupt RNA loops by reducing PTBP1 binding on pre-mRNAs to cause aberrant splicing in tumors. Our study presents a powerful method for exploring the functions of RBP-associated RNA-RNA proximal contacts in gene regulation and disease. Overall design: We developed a capture RIC-seq (CRIC-seq) method for the global mapping of RBP-associated in situ proximal RNA-RNA fragments. Two biological replicates of CRIC-seq libraries were generated for PTBP1 IP (+pCp), hnRNPA1 IP (+pCp), SRSF1 IP (+pCp), IgG IP (+pCp), PTBP1 IP (-pCp), hnRNPA1 IP (-pCp), and SRSF1 IP (-pCp) in HeLa cells. In addition, we also constructed two biological replicates of RIC-seq libraries for PTBP1 and hnRNPA1 knockdown cells to test the specificity of PTBP1- or hnRNPA1-associated RNA loops identified by CRIC-seq. To identify PTBP1 Cys23-mediated RNA loops and their influence on splicing regulation, we performed RNA-seq and CRIC-seq in the PTBP1-C23S mutant replaced (C23S+Dox) or not replaced (C23S-Dox) HeLa cells.