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

Abstract

Over recent years, long-range RNA structure has emerged as a factor that is fundamental to alternative splicing regulation. An increasing number of human disorders are now being associated with splicing defects; hence it is essential to develop methods that assess long-range RNA structure experimentally. RNA in situ conformation sequencing (RIC-seq) is a method that recapitulates RNA structure within physiological RNA-protein complexes. In this work, we juxtapose pairs of conserved complementary regions (PCCRs) that were predicted in silico with the results of RIC-seq experiments conducted in seven human cell lines. We show statistically that RIC-seq support of PCCRs correlates with their properties, such as equilibrium free energy, presence of compensatory substitutions, and occurrence of A-to-I RNA editing sites and forked eCLIP peaks. Exons enclosed in PCCRs that are supported by RIC-seq tend to have weaker splice sites and lower inclusion rates, which is indicative of post-transcriptional splicing regulation mediated by RNA structure. Based on these findings, we prioritize PCCRs according to their RIC-seq support and show, using antisense nucleotides and minigene mutagenesis, that PCCRs in two disease-associated human genes, PHF20L1 and CASK, and also PCCRs in their murine orthologs, 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.

Keywords: CASK; PCCR; PHF20L1; RIC-seq; RNA interaction; long-range; splicing.

FIGURE 1.

RNA contacts. (A) Digestion and religation of RNA strands adjacent to a PCCR result in RNA contacts (in the form of split reads) supporting the PCCR from the inner (2–3) or the outer (1–4) side. (B) The inner (I) and the outer (O) contacts correspond to the inner and outer arcs with respect to the RNA structure. The outer and the inner contacts are represented by neo-junctions and chimeric split reads, respectively.

FIGURE 2.

Properties of PCCRs in the IO category. The equilibrium free energy ΔG (A), the R-scape E-value (Rivas et al. 2017) (B), the frequency of A-to-I RNA editing sites (C), and the frequency of forked eCLIP peaks (D) near PCCRs supported inside and outside (IO category) in at least k cell lines. See Supplemental Figures S4 and S5 for the I and O categories, respectively. Statistically discernible differences at the 0.01% significance level and nonsignificant differences are denoted by (****) and “ns,” respectively (two-tailed Mann–Whitney test). Whiskers indicate 95% confidence intervals for proportions.

FIGURE 3.

Importance of RNA contacts near PCCRs. (A) For each PCCR, six 50-nt bins centering in the middle of the complementary sequence were chosen, and split read counts of RNA contacts from all RIC-seq experiments were computed for 6 × 6 = 36 combinations. (B) The performance (TPR, true positive rate, vs. FPR, false positive rate) of the random forest classifier predicting the presence of forked eCLIP peaks as a function of spread alone (green), RIC-seq support alone (orange), and spread and RIC-seq support together (blue). AUC is the area under the curve. (C) Feature importance (color map) for 36 bin combinations. The two most important features were the contacts between 1L and −1R and between −1L and 1R, which correspond to the inner and outer contacts immediately adjacent to the PCCR.

FIGURE 4.

Exons enclosed in PCCRs. (A) The average exon inclusion rate (Ψ) of exons in PCCRs that are supported inside and outside (IO category) in at least k cell lines. CDF represents cumulative probability density function. See also Supplemental Figure S6 for the I and O categories, respectively. (B) The distribution of r(Ψ, support), the Pearson correlation coefficient between exon inclusion rate Ψ and the number of reads supporting exon loop-out by PCCR (top), and the distribution of ΔΨ = Ψ_h − Ψ_l, where Ψ_h and Ψ_l are the average Ψ values in cell lines with and without RIC-seq support, respectively (bottom). Both distributions significantly depart from zero in the negative direction (Wilcoxon test, P-value <10⁻⁹). (C) The proportion of RIC-seq-supported cases among exons looped out by a PCCR, for PTC-containing (PTC+) exons and cassette exons without PTC (PTC−). Whiskers indicate 95% confidence intervals for proportions. (D) The equilibrium free energy (ΔG) of PCCRs looping-out PTC+ and PTC− exons in the supported and unsupported groups. Statistically discernible differences at the 1% significance level and nonsignificant differences are denoted by ** and “ns,” respectively (one-tailed Mann–Whitney test).

FIGURE 5.

Case study of PHF20L1. (A) Genomic organization of the cassette exon 6, the complementary sequences R1 and R2, and their respective AONs (AON1 and AON2). (B) The treatment with AON1 or AON2 almost completely suppresses exon 6 skipping; NT (nontreated control); C (control AON); neg (negative control). (C) The scheme of a minigene expressing a fragment of the PHF20L1 gene. Primer locations are indicated by the arrows. (D) Mutagenesis in the minigene. In m1 and m2 mutants, the base pairing between R1 and R2 is disrupted by sequence reversal; in the compensatory double mutant m1m2, it is restored. (E) Exon 6 skipping in m1 and m2 mutants is suppressed, but in m1m2 it returns to that of the WT. Statistically discernible differences at the 1%, and 0.1% significance level are denoted by **, and ***, respectively. Nonsignificant differences are denoted by “ns.”

FIGURE 6.

Case study of CASK. (A) Genomic organization of the cassette exon 19, complementary sequences R3 and R4, and their respective AONs (AON3 and AON4). (B) The treatment with AON3 or AON4 significantly reduces exon 19 skipping; NT (nontreated control); C (control AON); neg (negative control). (C) The scheme of a minigene expressing a fragment of the CASK gene. (D) In m3 and m4 mutants, the base pairing between R3 and R4 is disrupted by sequence reversal; in the compensatory double mutant m3m4, it is restored. (E) Exon 19 skipping in m3 and m4 is suppressed, but in m3m4 it returns to that of the WT. Statistically discernible differences at the 5%, 1%, and 0.1% significance level are denoted by *, **, and ***, respectively. Nonsignificant differences are denoted by “ns.”

Sergei Margasyuk