Biology subjects

Pervouchine, D.

Publications and source records attributed to Pervouchine, D..

8 recordsLinked to original sources

bioRxiv2025

A comparative study of genital lichen sclerosus transcriptomes

Genital lichen sclerosus (GLS) is a chronic inflammatory dermatosis that affects the genital skin. Despite different clinical manifestations, the pathogenesis of GLS in men and women is thought to be common and is attributed to a combination of autoimmune and genetic factors. In this study, we compared the transcriptomic profiles of penile (mGLS) and vulvar lichen sclerosus (VLS) with the objective to identify commonly deregulated genes. We observed a substantial heterogeneity of the transcriptomic signatures in mGLS samples which is driven by different compositions of immune infiltrates. In mGLS, gene expression signatures strongly indicate epidermis dysfunction and overexpression of epithelial inflammation marker Keratin 6 (KRT6) and chitinase CHIT1. No significant changes in the expression levels of known GLS markers such as VIM, CTNNB1, LGALS7 and ECM1 were detected, however, changes in the expression levels of genes associated with autoimmune diseases and genes upregulated in squamous cell carcinoma were observed, including TNF, CCNB1 and RUNX3. There was no enrichment in HCV-derived polyU/UC insertions that were reported previously. Instead, we have identified a long non-coding RNA DRAIC with a large coding potential that is commonly upregulated in mGLS and VLS. Together, our results represent a comprehensive catalog of shared transcriptomic signatures including novel biomarkers and potential therapeutic targets.

bioinformatics↗

bioRxiv2025

Ancestral intronic splicing regulatory elements in the SCNα gene family

SCN genes encode components of voltage-gated sodium channels that are crucial for generating electrical signals. Humans have ten paralogous SCN genes, some of which contain duplicated mutually exclusive exons 5a and 5b. In reconstructing their evolutionary history, we found multiple unannotated copies of exon 5 in distant species and showed that exon 5 duplication goes back to a common ancestor of the SCN gene family. We char-acterized splicing patterns of exons 5a and 5b across tissues, tumors, and developmental stages, and demonstrated that the nonsense mediated decay (NMD) system is not the ma-jor factor contributing to their mutually exclusive choice. Comparison of SCN2A, SCN3A, SCN5A, and SCN9A intronic nucleotide sequences revealed multiple Rbfox2 binding sites and two highly conserved intronic splicing regulatory elements (ISRE) that are shared be-tween paralogs. Minigene mutagenesis and blockage by antisense oligonucleotides showed that the formation of RNA structure between ISRE promotes exon 5b skipping in SCN9A. The inclusion of exon 5b is also suppressed in siRNA-mediated knockdown of Rbfox2, which makes the collective action of RNA structure and Rbfox2 compatible with the model of a structural RNA bridge. ISRE sequences are conserved from human to elephant shark and may represent an ancient, evolutionarily conserved regulatory mechanism. Our results demonstrate the power of comparative sequences analysis in application to paralogs for elucidating splicing regulatory programs.

bioinformatics↗

bioRxiv2023

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

The mammalian BRD2 and BRD3 genes encode structurally related proteins from the bromodomain and extraterminal domain (BET) protein family. The expression of BRD2 is regulated by unproductive splicing upon inclusion of exon 3b, which is located in the region encoding a bromodomain. Bioinformatic analysis indicated that BRD2 exon 3b inclusion is controlled by a pair of conserved complementary regions (PCCR) located in the flanking introns. Furthermore, we identified a highly conserved element encoding a cryptic poison exon 5b and a previously unknown PCCR in the intron between exons 5 and 6 of BRD3, however outside of the homologous bromodomain. Minigene mutagenesis and blockage of RNA structure by antisense oligonucleotides demonstrated that RNA structure controls the rate of inclusion of poison exons. The patterns of BRD2 and BRD3 expression and splicing show downregulation upon inclusion of poison exons, which become skipped in response to transcription elongation slowdown, further confirming a role of PCCRs in unproductive splicing regulation. We conclude that BRD2 and BRD3 independently acquired poison exons and RNA structures to dynamically control unproductive splicing. This study describes a convergent evolution of regulatory unproductive splicing mechanisms in these genes providing implications for selective modulation of their expression in therapeutic applications.

bioinformatics↗

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↗

bioRxiv2020

An extended catalogue of tandem alternative splice sites in human tissue transcriptomes

Tandem alternative splice sites (TASS) is a special class of alternative splicing events that are characterized by a close tandem arrangement of splice sites. Most TASS lack functional characterization and are believed to arise from splicing noise. Based on the RNA-seq data from the Genotype Tissue Expression project, we present an extended catalogue of TASS in healthy human tissues and analyze their tissue-specific expression. The expression of TASS is usually dominated by one major splice site (maSS), while the expression of minor splice sites (miSS) is at least an order of magnitude lower. Among 73k miSS with sufficient read support, 12k (17%) are significantly expressed above the expected noise level, and among them 2k are expressed tissue-specifically. We found significant correlations between tissue-specific expression of RNA-binding proteins (RBP) and tissue-specific expression of miSS that is consistent with miSS response to RBP inactivation by shRNA. In combination with RBP profiling by eCLIP, this allowed prediction of novel cases of tissue-specific splicing regulation including a miSS in QKI mRNA that is likely regulated by PTBP1. According to the structural annotation of the human proteome, tissue-specific miSS are enriched within disordered regions, and indels induced by miSS are enriched with short linear motifs and post-translational modification sites. Nonetheless, more than 15% of tissue-specific miSS affect structured protein regions and may adjust protein-protein interactions or modify the stability of the protein core. The significantly expressed miSS evolve under the same selection pressure as maSS, while other miSS lack signatures of evolutionary selection and conservation. Using mixture models, we estimated that not more than 10% of maSS and not more than 50% of significantly expressed miSS are noisy, while the proportion of noisy splice sites among not significantly expressed miSS is above 70%.

bioinformatics↗

bioRxiv2020

Multiple competing RNA structures dynamically control alternative splicing in human ATE1 gene

The mammalian Ate1 gene encodes an arginyl transferase enzyme, which is essential for embryogenesis, male meiosis, and regulation of the cytoskeleton. Reduced levels of Ate1 are associated with malignant transformations and serve as a prognostic indicator of prostate cancer metastasis. The tumor suppressor function of Ate1 depends on the inclusion of one of the two mutually exclusive exons (MXE), exons 7a and 7b. Here, we report that the molecular mechanism underlying MXE splicing in Ate1 involves five conserved regulatory intronic elements R1-R5, of which R1 and R4 compete for base pairing with R3, while R2 and R5 form an ultra-long-range RNA structure spanning 30 Kb. In minigenes, single and double mutations that disrupt base pairings in R1R3 and R3R4 lead to the loss of MXE splicing, while compensatory triple mutations that restore the RNA structure also revert splicing to that of the wild type. Blocking the competing base pairings by locked nucleic acid (LNA)/DNA mixmers complementary to R3 leads to the loss of MXE splicing, while the disruption of the ultra-long-range R2R5 interaction changes the ratio of mutually exclusive isoforms in the endogenous Ate1 pre-mRNA. The upstream exon 7a becomes more included than the downstream exon 7b in response to RNA Pol II slowdown, however it fails to do so when the ultra-long-range R2R5 interaction is disrupted. In sum, we demonstrated that mutually exclusive splicing in Ate1 is controlled by two independent, dynamically interacting and functionally distinct RNA structure modules. The molecular mechanism proposed here opens new horizons for the development of therapeutic solutions, including antisense correction of splicing.

molecular biology↗

bioRxiv2019

Systematic re-annotation of 191 genes associated with early-onset epilepsy unmasks de novo variants linked to Dravet syndrome in novel SCN1A exons

The early infantile epileptic encephalopathies (EIEE) are a group of rare, severe neurodevelopmental disorders, where even the most thorough sequencing studies leave 60-65% of patients without a molecular diagnosis. Here, we explore the incompleteness of transcript models used for exome and genome analysis as one potential explanation for lack of current diagnoses. Therefore, we have updated the GENCODE gene annotation for 191 epilepsy-associated genes, using human brain-derived transcriptomic libraries and other data to build 3,550 novel putative transcript models. The extended transcriptional footprint of these genes allowed for 294 intronic or intergenic variants, found in human mutation databases, to be reclassified as exonic, while a further 70 intronic variants were reclassified as splice-site proximal. Using SCN1A as a case study due to its close phenotype/genotype correlation with Dravet syndrome, we screened 122 people with Dravet syndrome, or a similar phenotype, with a panel of novel exon sequences representing eight established genes and identified two de novo SCN1A variants that now, through improved gene annotation can be ascribed to residing among novel exons. These two (from 122 screened patients, 1.6%) new molecular diagnoses carry significant clinical implications. Furthermore, we identified a previously-classified SCN1A intronic Dravet-associated variant that now lies within a deeply conserved novel exon. Our findings illustrate the potential gains of thorough gene annotation in improving diagnostic yields for genetic disorders. We would expect to find new molecular diagnoses in our 191 genes that were originally suspected by clinicians for patients, with a negative diagnosis.

genomics↗

bioRxiv2018

Novel autoregulatory cases of alternative splicing coupled with nonsense-mediated mRNA decay

Nonsense-mediated decay (NMD) is a eukaryotic mRNA surveillance system that selectively degrades transcripts with premature termination codons (PTC). Many RNA-binding proteins (RBP) regulate their expression levels by a negative feedback loop, in which RBP binds its own pre-mRNA and causes alternative splicing to introduce a PTC. We present a bioinformatic framework to identify novel such autoregulatory feedback loops by combining eCLIP assays for a large panel of RBPs with the data on shRNA inactivation of NMD pathway, and shRNA-depletion of RBPs followed by RNA-seq. We show that RBPs frequently bind their own pre-mRNAs and respond prominently to NMD pathway disruption. Poison and essential exons, i.e., exons that trigger NMD when included in the mRNA or skipped, respectively, respond oppositely to the inactivation of NMD pathway and to the depletion of their host genes, which allows identification of novel autoregulatory mechanisms for a number of human RBPs. For example, SRSF7 binds its own pre-mRNA and facilitates the inclusion of two poison exons; SFPQ binding promotes switching to an alternative distal 3-UTR that is targeted by NMD; RPS3 activates a poison 5-splice site in its pre-mRNA that leads to a frame shift; U2AF1 binding activates one of its two mutually exclusive exons, leading to NMD; TBRG4 is regulated by cluster splicing of its two essential exons. Our results indicate that autoregulatory negative feedback loop of alternative splicing and NMD is a generic form of post-transcriptional control of gene expression.

bioinformatics↗