RNAin situconformation sequencing reveals novel long-range RNA structures with impact on splicing

Abstract

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.

BRD2 and BRD3 genes independently evolved RNA structures to control unproductive splicing

“…To test the effect of RNA structure on the inclusion of poison exons in BRD2 and BRD3 , we followed the strategy outlined previously [29, 30]. Namely, we measure splicing changes in the endogenous pre-mRNA in response to the blockage of PCCRs by antisense oligonucleotides (AONs), and construct minigenes carrying mutated gene fragments to assess splicing outcomes in single and double mutants.…”

“…In recent years, experimental and computational studies have demonstrated that RNA structure is critically important for alternative splicing regulation [35,29,26]. While the overall function of alternative splicing can arguably be attributed to expanding the proteome diversity [36,37,38], the functional implication of altering the primary sequence of a particular protein in most cases remains undocumented, thereby limiting the interpretation of RNA structure's impact.…”

“…TPM (transcripts per million) values were calculated from the read coverage using RNAnorm package version 2.0.0. Split read counts and percent-spliced-in metrics were computed by IPSA pipeline [54] with default settings as before [29]. Namely, the exon inclusion rate (Ψ) was computed as, where inc is the number of split reads supporting exon inclusion, and exc is the number of split reads supporting exon exclusion.…”

“…RT-PCR analysis was used to assess the ratio of splice isoforms as described elsewhere [29]. qPCR reactions were performed in triplicates in a final volume of 12µl in 96-well plates with 420 nM gene-specific primers, 2µl of cDNA using 5X qPCRmix-HS SYBR reaction mix (Evrogen).…”