Biology subjects

Margasyuk, S.

Publications and source records attributed to Margasyuk, S..

4 recordsLinked to original sources

bioRxiv2022

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.

molecular biology↗

bioRxiv2022

RNAcontacts, a pipeline for predicting contacts from RNA proximity ligation assays

BackgroundHigh-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. FindingsHere, 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. ConclusionsRNAcontacts 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 at https://github.com/smargasyuk/RNAcontacts/

bioinformatics↗

bioRxiv2022

Tissue-specific regulation of gene expression via unproductive splicing

Eukaryotic gene expression is regulated post-transcriptionally by a mechanism called unproductive splicing, in which mRNA is triggered to degradation by the nonsense-mediated decay (NMD) pathway as a result of regulated alternative splicing (AS). Only a few dozen unproductive splicing events (USEs) are currently documented, and many more remain to be identified. Here, we analyzed RNA-seq experiments from the Genotype-Tissue Expression (GTEx) Consortium to identify USEs, in which an increase in the NMD isoform splicing rate is accompanied by tissue-specific downregulation of the host gene. Further, to characterize RBPs that regulate USEs, we superimposed these results with RNA-binding protein (RBP) footprinting data and experiments on the response of the transcriptome to the perturbation of expression of a large panel of RBPs. Concordant tissue-specific changes between the expression of RBP and USE splicing rate revealed a high-confidence regulatory network including 27 tissue-specific USEs with strong evidence of RBP binding. Among them, we found previously unknown PTBP1-controlled events in the DCLK2 and IQGAP1 genes, for which we confirmed the regulatory effect using siRNA-knockdown experiments in the A549 cell line. In sum, we present a transcriptomic pipeline that allows the identification of tissue-specific USEs, potentially many more than we have reported here using stringent filters.

bioinformatics↗

bioRxiv2022

Transcriptome sequencing suggests that pre-mRNA splicing counteracts premature intronic polyadenylation

Alternative splicing (AS) and alternative polyadenylation (APA) are two crucial steps in the post-transcriptional regulation of eukaryotic gene expression. Protocols capturing and sequencing RNA 3-ends have uncovered widespread intronic polyadenylation (IPA) in normal and disease conditions, where it is currently attributed to stochastic variations in the pre-mRNA processing. Here, we took advantage of the massive amount of RNA-seq data generated by the Genotype Tissue Expression project (GTEx) to simultaneously identify and match tissue-specific expression of intronic polyadenylation sites with tissue-specific splicing. A combination of computational methods including the analysis of short reads with non-templated adenines revealed that APA events are more abundant in introns than in exons. While the rate of IPA in composite terminal exons and skipped terminal exons expectedly correlates with splicing, we observed a considerable fraction of IPA events that lack AS support and attributed them to spliced polyadenylated introns (SPI). We hypothesize that SPIs represent transient byproducts of a dynamic coupling between APA and AS, in which the spliceosome removes an intron after cleavage and polyadenylation have already occurred in it. These findings indicate that cotranscriptional pre-mRNA splicing could serve as a rescue mechanism to suppress premature transcription termination at intronic polyadenylation sites.

bioinformatics↗