High-throughput RNA proximity ligation assays are molecular methods that simultaneously analyze spatial proximity of many RNAs in living cells. Their principle is based on cross-linking, fragmentation, and consequent religation of RNAs followed by high-throughput sequencing. The generated fragments have two distinct types of splits, one resulting from pre-mRNA splicing, and the other resulting from ligating spatially close RNA strands. Here, we present RNAcontacts, a universal pipeline for detecting RNA-RNA contacts in high-throughput RNA proximity ligation assays. It circumvents the inherent problem of mapping sequences with two distinct split types using a two-pass alignment, in which splice junctions are inferred from a control RNA-seq experiment on the first pass and then provided to the aligner on the second pass as bona fide introns. This approach allows for a more sensitive detection of RNA contacts and has higher specificity with respect to splice junctions that are present in the biological sample in comparison to previously developed methods. RNAcontacts extracts contacts, clusters their ligation points, computes the read support, and generates tracks for the visualization through the UCSC Genome Browser. It is implemented in a reproducible and scalable workflow management system Snakemake that allows fast and uniform processing of multiple datasets. RNAcontacts represents a generic pipeline for the detection of RNA contacts that can be used with any proximity ligation method as long as one of the interacting partners is RNA. RNAcontacts is available via github repository https://github.com/smargasyuk/RNAcontacts.
Over past 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.
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.
High-throughput RNA proximity ligation assays are molecular methods that are used to simultaneously analyze the spatial proximity of many RNAs in living cells. Their principle is based on cross-linking, fragmentation, and subsequent religation of RNAs, followed by high-throughput sequencing. The generated fragments have two different types of splits, one resulting from pre-mRNA splicing and the other formed by the ligation of spatially close RNA strands. Here, we present RNAcontacts, a universal pipeline for detecting RNARNA contacts in high-throughput RNA proximity ligation assays. RNAcontacts circumvents the inherent problem of mapping sequences with two distinct types of splits using a two-pass alignment, in which splice junctions are inferred from a control RNA-seq experiment on the first pass and then provided to the aligner as bona fide introns on the second pass. Compared to previously developed methods, our approach allows for a more sensitive detection of RNA contacts and has a higher specificity with respect to splice junctions that are present in the biological sample. RNAcontacts automatically extracts contacts, clusters their ligation points, computes the read support, and generates tracks for visualizing through the UCSC Genome Browser. The pipeline is implemented in Snakemake, a reproducible and scalable workflow management system for rapid and uniform processing of multiple datasets. RNAcontacts is a generic pipeline for the detection of RNA contacts that can be used with any proximity ligation method as long as one of the interacting partners is RNA. RNAcontacts is available via the GitHub repository https://github.com/smargasyuk/RNAcontacts/
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