Biology subjects

Mironov, A. A.

Publications and source records attributed to Mironov, A. A..

14 recordsLinked to original sources

bioRxiv2025

Global changes in unproductive splicing and the NMD system efficiency in tumors

The Nonsense-Mediated Decay (NMD) pathway is a mRNA quality control mechanism, which not only degrades deleterious transcripts but also orchestrates a large number of post-transcriptional regulatory programs through unproductive splicing. Here, we developed a robust metric derived from the quantification of splicing in the RNA-seq data to measure NMD efficiency at a sample level. We demonstrate that NMD efficiency varies substantially both between and within tissues, with the magnitude of variation comparable to that observed upon knockdown of the core NMD factor UPF1. By analyzing TCGA cancer cohorts, we further show that, in many tumors, unproductive splicing events exhibit coordinated changes towards either collective suppression or collective activation of NMD isoforms, which is indicative of a global deregulation of the NMD pathway activity. Consistently, we observed a striking divergence of NMD efficiency in cancers from the tissue-specific baseline level suggesting that tumors partially erase the NMD signature of their tissue of origin. The application of the developed metric to RNA-binding protein knockdowns allowed identification of several novel potential regulators of NMD efficiency. In sum, this study provides a solid framework for NMD efficiency quantification, describes its biological and clinical relevance, and opens new avenues for dissecting mechanisms of post-transcriptional gene expression regulation by the NMD pathway.

bioinformatics↗

bioRxiv2024

Joint analysis of RNA-DNA and DNA-DNA interactomes reveals their strong association

At the moment, many non-coding RNAs that perform a variety of functions in the regulation of chromatin processes are known. An increasing number of protocols allow researchers to study RNA-DNA interactions and shed light on new aspects of the RNA-chromatin interactome. The Hi-C protocol, which enables the study of chromatins three-dimensional organization, has already led to numerous discoveries in the field of genome 3D organization. We conducted a comprehensive joint analysis of the RNA-DNA interactome and chromatin structure across different human and mouse cell lines. We have shown that these two phenomena are closely related in many respects, with the nature of this relationship being both tissue-specific and conserved across human and mouse.

bioinformatics↗

bioRxiv2024

Comprehensive analysis of RNA-chromatin, RNA- and DNA-protein interactions

RNA-chromatin interactome data is considered to be one of the noisiest types of data in biology. This is due to protein-coding RNA contacts and non-specific interactions between RNA and chromatin caused by protocol specifics. Therefore, finding regulatory interactions between certain transcripts and genome loci requires a wide range of filtering techniques to obtain significant results. Using data on pairwise interactions between these molecules, we propose a concept of triad interaction involving RNA, protein and a DNA locus. The constructed triads show significantly less noise contacts and are more significant when compared to a background model for generating pairwise interactions. RNA-chromatin contacts data can be used to validate the proposed triad object as positive (Red-ChIP experiment) or negative (RADICL-Seq NPM) controls. Our approach also filters RNA-chromatin contacts in chromatin regions associated with protein functions based on ChromHMM annotation.

bioinformatics↗

bioRxiv2023

BaRDIC: robust peak calling for RNA-DNA interaction data

Chromatin-associated non-coding RNAs play important roles in various cellular processes by targeting genomic loci. Two types of genome-wide NGS experiments exist to detect such targets: "one-to-all", which focuses on targets of a single RNA, and "all-to-all", which captures targets of all RNAs in a sample. As with many NGS experiments, they are prone to biases and noise, so it becomes essential to detect "peaks" - specific interactions of an RNA with genomic targets. Here we present BaRDIC - Binomial RNA-DNA Interaction Caller - a tailored method to detect peaks in both types of RNA-DNA interaction data. BaRDIC is the first tool to simultaneously take into account the two most prominent biases in the data: chromatin heterogeneity and distance-dependent decay of interaction frequency. Since RNAs differ in their interaction preferences, BaRDIC adapts peak sizes according to the abundances and contact patterns of individual RNAs. These features enable BaRDIC to make more robust predictions than currently applied peak-calling algorithms and better handle the characteristic sparsity of all-to-all data. BaRDIC package is freely available at https://github.com/dmitrymyl/BaRDIC.

bioinformatics↗

bioRxiv2023

Foreign RNA spike-ins enable accurate allele-specific expression analysis at scale

MotivationAnalysis of allele-specific expression is strongly affected by the technical noise present in RNA-seq experiments. Previously, we showed that technical replicates can be used for precise estimates of this noise, and we provided a tool for correction of technical noise in allele-specific expression analysis. This approach is very accurate but costly due to the need for two or more replicates of each library. Here, we develop a spike-in approach that is highly accurate at only a small fraction of the cost. ResultsWe show that a distinct RNA added as a spike-in before library preparation reflects technical noise of the whole library and can be used in large batches of samples. We experimentally demonstrate the effectiveness of this approach using combinations of RNA from species distinguishable by alignment, namely, mouse, human, and C.elegans. Our new approach, controlFreq, enables highly accurate and computationally efficient analysis of allele-specific expression in (and between) arbitrarily large studies at an overall cost increase of ~ 5%. AvailabilityAnalysis pipeline for this approach is available at GitHub as R package controlFreq (github.com/gimelbrantlab/controlFreq). Contactagimelbrant@altius.org

bioinformatics↗

bioRxiv2022

Vimentin intermediate filaments provide structural stability to the mammalian Golgi apparatus

The Golgi apparatus comprises a connected ribbon of stacked cisternal membranes localized to the perinuclear region of most vertebrate cells. The position and morphology of this organelle depends upon interactions with microtubules and the actin cytoskeleton. In contrast, we know relatively little about the relationship of the Golgi apparatus with intermediate filaments. In this study we show that the Golgi is in close physical proximity to vimentin intermediate filaments (IFs) in cultured mouse and human cells. We also show that the trans-Golgi network coiled-coil protein GORAB can physically associate with IFs. Although loss of vimentin and/or GORAB does not have major effects upon Golgi morphology at steady-state, the Golgi undergoes more rapid disassembly upon chemical disruption with the drug brefeldin A, and slower reassembly upon drug washout, in vimentin knockout cells. Moreover, loss of vimentin causes reduced Golgi ribbon integrity when cells are cultured on high stiffness hydrogels, which is exacerbated by loss of GORAB. These results indicate that vimentin IFs contribute to the structural stability of the Golgi apparatus, and suggest a role for GORAB in this process.

cell biology↗

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

PIP4K2B is a mechanosensor and induces heterochromatin-driven nuclear softening through UHRF1.

Phosphatidylinositol-5-phosphate (PtdIns5P)-4-kinases (PIP4Ks) are stress-regulated phosphoinositide kinases able to phosphorylate PtdIns5P to PtdIns(4, 5)P2. In cancer patients their expression is typically associated with bad prognosis. Among the three PIP4K isoforms expressed in mammalian cells, PIP4K2B is the one with more prominent nuclear localization. Here, we unveil the role for PIP4K2B as mechanosensor. PIP4K2B protein level, indeed, strongly decreases in cells growing on soft substrates. Its direct silencing or pharmacological inhibition, mimicking cell response to soft, triggers a concomitant reduction of the epigenetic regulator UHRF1 and induces changes in nuclear polarity, nuclear envelope tension and chromatin compaction. This substantial rewiring of the nucleus mechanical state drives YAP cytoplasmic retention and impairment of its activity as transcriptional regulator, finally leading to defects in cell spreading and motility. Since YAP signalling is essential for initiation and growth of human malignancies, our data suggest that potential therapeutic approaches targeting PIP4K2B could be beneficial in the control of the altered mechanical properties of cancer cells.

cell biology↗

bioRxiv2021

ortho2align: a sensitive approach for searching for orthologues of novel lncRNAs

BackgroundMany novel long noncoding RNAs have been discovered in recent years due to advances in high-throughput sequencing experiments. Finding orthologues of these novel lncRNAs might facilitate clarification of their functional role in living organisms. However, lncRNAs exhibit low sequence conservation, so specific methods for enhancing the signal-to-noise ratio were developed. Nevertheless, current methods such as transcriptomes comparison approaches or searches for conserved secondary structures are not applicable to novel lncRNAs dy design. ResultsWe present ortho2align -- a versatile sensitive synteny-based lncRNA orthologue search tool with statistical assessment of sequence conservation. This tool allows control of the specificity of the search process and optional annotation of found orthologues. ortho2align shows similar performance in terms of sensitivity and resource usage as the state-of-the-art method for aligning orthologous lncRNAs but also enables scientists to predict unannotated orthologous sequences for lncRNAs in question. Using ortho2align, we predicted orthologues of three distinct classes of novel human lncRNAs in six Vertebrata species to estimate their degree of conservation. ConclusionsBeing designed for the discovery of unannotated orthologues of novel lncRNAs in distant species, ortho2align is a versatile tool applicable to any genomic regions, especially weakly conserved ones. A small amount of input files makes ortho2align easy to use in orthology studies as a single tool or in bundle with other steps that researchers will consider sensible.

bioinformatics↗

bioRxiv2020

The Step-Wise C-Truncation and Transport of ESyt3 to Lipid Droplets Reveals a Mother Primordial Cisterna

Extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER) proteins consisting of an SMP domain and multiple C2 domains that bind phospholipids and Ca2+. E-Syts create contact junctions between the ER and plasma membrane to facilitate lipid exchange. During adipocyte differentiation, the proteasome-based removal of the C2C domain results in targeting of E-Syt3 to the primordial cisterna, a previously undescribed giant annular organelle that mothers the LDs. Further cleavage causes the E-Syt3 relocation to the surface of LDs. Fragmentation of the primordial cisterna and LD budding into its lumen are early events in the biogenesis of LDs in the 3T3-L1 adipocyte. Electron tomography-based 3D reconstruction of the fragmented primordial cisterna revealed patches of a tightly packed E-Syt3-rich network of varicose tubules in close contact with young LDs. Esyt3 binds avidly phosphatidylethanolamine through its SMP domain, a main component of the LD membrane that fosters LD biogenesis. Repression of E-Syt3 effectively inhibits LD biogenesis and growth.

cell biology↗

bioRxiv2020

Unexpected variability of allelic imbalance estimates from RNA sequencing

RNA sequencing and other experimental methods that produce large amounts of data are increasingly dominant in molecular biology. However, the noise properties of these techniques have not been fully understood. We assessed the reproducibility of allele-specific expression measurements by conducting replicate sequencing experiments from the same RNA sample. Surprisingly, variation in the estimates of allelic imbalance (AI) between technical replicates was up to 7-fold higher than expected from commonly applied noise models. We show that AI overdispersion varies substantially between replicates and between experimental series, appears to arise during the construction of sequencing libraries, and can be measured by comparing technical replicates. We demonstrate that compensation for AI overdispersion greatly reduces technical variation and enables reliable differential analysis of allele-specific expression across samples and across experiments. Conversely, not taking AI overdispersion into account can lead to a substantial number of false positives in analysis of allele-specific gene expression

bioinformatics↗

bioRxiv2020

Origin of the nuclear proteome on the basis of pre-existing nuclear localization signals in prokaryotic proteins

The origin of the selective nuclear protein import machinery, which consists of nuclear pore complexes and adaptor molecules interacting with the nuclear localization signals (NLSs) of cargo molecules, was one of the most important events in the evolution of the eukaryotic cell. How the proteins were selected for import into the forming nuclei remains an open question. Here, we demonstrate that functional NLSs may be integrated inside nucleotide-binding domains of both eukaryotic and prokaryotic proteins and may co-evolve with these domains. We propose that the pre-existence of NLSs inside prokaryotic proteins dictated, at least partially, the nuclear proteome composition.

evolutionary biology↗

bioRxiv2019

Studying RNA#8211;DNA interactome by Red-C identifies noncoding RNAs associated with repressed chromatin compartment and reveals transcription dynamics

Non-coding RNAs (ncRNAs) participate in various biological processes, including regulating transcription and sustaining genome 3D organization. Here, we present a method termed Red-C that exploits proximity ligation to identify contacts with the genome for all RNA molecules present in the nucleus. Using Red-C, we uncovered the RNA-DNA interactome of human K562 cells and identified hundreds of ncRNAs enriched in active or repressed chromatin, including previously undescribed RNAs. We found two microRNAs--MIR3648 and MIR3687 transcribed from the rRNA locus--that are associated with inactive chromatin genome wide. These miRNAs favor bulk heterochromatin over Polycomb-repressed chromatin and interact preferentially with late-replicating genomic regions. Analysis of the RNA-DNA interactome also allowed us to trace the kinetics of messenger RNA production. Our data support the model of co-transcriptional intron splicing, but not the hypothesis of the circularization of actively transcribed genes.

genomics↗

bioRxiv2019

Uncoating of COPII from ER exit site membranes precedes cargo accumulation and membrane fission

COPII and COPI are considered to be analogous sets of vesicle coat protein heterocomplexes. Coupled to cargo selection, they mediate the formation of membrane vesicles translocating in opposite directions to differ rent destinations within the secretory pathway. Here, live cell and electron microscopy provided evidence for a different localization and mode of function of the COPII coat during protein export from the endoplasmic reticulum (ER). Pharmaceutical and genetic perturbations of ER-Golgi transport were used to demonstrate that COPII is recruited to membranes defining the boundary of ER-ER Exit Sites (ERES) where it facilitates selective cargo concentration. Uncoating of COPII membranes precedes cargo accumulation and fission of Golgi-bound carriers. Moreover, we report what may be direct transfer of cargo to the Golgi apparatus from Golgi-associated BFA sensitive ERESs. Finally, in ldlF cells the stably expressed functional {varepsilon}-COPI-EYFP labeled both ERESs and anterograde carriers. These findings change our understanding of the role of coat proteins in ER to Golgi transport.

cell biology↗