RNA in situ conformation sequencing reveals novel long-range RNA structures with impact on splicing

Over past years, long-range RNA structure has emerged as a factor that is fundamental to alternative splicing regulation. Since an increasing number of human disorders are now being associated with splicing defects, it is essential to develop methods that assess long-range RNA structure experimentally. RNA in situ conformation sequencing (RIC-seq) is the method that recapitulates RNA structure within physiological RNA-protein complexes. In this work, we juxtapose RIC-seq experiments conducted in eight human cell lines with pairs of conserved complementary regions (PCCRs) that were predicted in silico. We show statistically that RIC-seq support strongly correlates with PCCR properties such as equilibrium free energy, presence of compensatory substitutions, and occurrence of A-to-I RNA editing sites and forked eCLIP peaks. Based on these findings, we prioritize PCCRs according to their RIC-seq support and show experimentally using antisense nucleotides and minigene mutagenesis that PCCRs in two disease-associated genes, PHF20L1 and CASK, impact alternative splicing. In sum, we demonstrate how RIC-seq experiments can be used to discover functional long-range RNA structures, and particularly those that regulate alternative splicing.