Biology subjects

Dmitriev, S. E.

Publications and source records attributed to Dmitriev, S. E..

17 recordsLinked to original sources

bioRxiv2026

Heterogeneous epigenetic regulatory patterns link mammalian aging, development, and mortality

Aging is often described as a monotonic accumulation of cellular damage, yet all-cause mortality follows a U-shaped trajectory with age, suggesting non-monotonic molecular changes. We investigated links between early childhood development, aging, and chronic diseases by analyzing DNA methylation in mammalian blood. A meta-analysis of 16 human chronic diseases revealed heterogeneous methylation signatures that formed 2 major disease clusters distinguished by their associations with development and sex-related methylation changes. Although epigenetic entropy increased monotonically across the lifespan, several diseases reduced blood DNA methylation entropy independently of blood cell composition. Across mammals, many CpG sites, particularly in intergenic regions, followed U-shaped age-related methylation changes that paralleled mortality curves. Based on these patterns, we developed epigenetic clocks that predict expected mortality across species and tissues and are effective in detecting a range of disease models. Overall, our findings reveal fundamental links between epigenetic regulation during development, aging, and chronic diseases.

bioinformatics↗

bioRxiv2026

The conserved β-hairpin of the SUI1 domain is a dual-function structural module governing translation initiation and ribosome recycling in yeast

Most eukaryotic mRNAs encode a single functional polypeptide. Following translation termination, both the large and small ribosomal subunits are typically released from the mRNA by ribosome recycling factors. However, after translating short upstream open reading frames (uORFs) within the 5' untranslated regions (UTRs), ribosomes can remain associated with the mRNA and reinitiate translation. This process is regulated by the heterodimer MCTS1{middle dot}DENR (Tma20p{middle dot}Tma22p in yeast). DENR/Tma22p harbors a SUI1 domain, structurally homologous to the translation initiation factor eIF1/Sui1p, which features a conserved, positively charged {beta}-hairpin loop critical for eIF1 function. Despite this structural similarity, the functional significance of specific elements within DENR/Tma22p remains unexplored. Here, we used in vivo reporter assays in Saccharomyces cerevisiae to quantify reinitiation efficiency following translation of either a short uORF (in the 5' UTR) or a full-length coding sequence (in the 3' UTR). Systematic analysis of single, double, and triple deletions of TMA20, TMA22, and TMA64 (a homolog of Tma20p{middle dot}Tma22p) revealed that the Tma20p{middle dot}Tma22p complex exerts a dominant role over Tma64p in modulating reinitiation, while exhibiting functional interplay between the two factors. Using knockout strains complemented with Tma22p variants, we further demonstrated that the positively charged residues of the {beta}-hairpin loop 1 are essential for Tma22p recycling activity. Unexpectedly, deletion of the entire SUI1 domain was less deleterious, and eIF1/Sui1p was able to partially substitute for the SUI1 domain of Tma22p within a chimeric protein context. Our findings establish the {beta}-hairpin loop 1 of the DENR/Tma22p SUI1 domain as a critical determinant for ribosome recycling and reinitiation, and raise the question of whether MCTS1/Tma20p can promiscuously operate with either DENR/Tma22p or eIF1/Sui1p - two specialized factors that evolved from a common structural scaffold to govern distinct steps in the translation cycle.

molecular biology↗

bioRxiv2025

The p48 isoform of the PA2G4/EBP1/ITAF45 oncoprotein is required for the encephalomyocarditis virus IRES-driven translation initiation

Internal ribosome entry sites (IRESs) enable cap-independent initiation of picornaviral RNA translation and, together with canonical translation initiation factors, typically require specific cellular proteins known as IRES trans-acting factors (ITAFs). While the type II IRES of foot-and-mouth disease virus (FMDV, an aphthovirus) has been shown to depend on the oncoprotein ITAF45, also known as Proliferation-Associated 2G4 (PA2G4) or ErbB-3 receptor Binding Protein (EBP1), for in vitro assembly of the 48S pre-initiation complex, some related type II IRESs, such as that of encephalomyocarditis virus (EMCV, a cardiovirus), can form the initiation complex independently of ITAF45. In this study, we performed a genome-wide CRISPR screen and identified knockouts of PA2G4/EBP1/ITAF45 in cells that survive EMCV infection, suggesting an important role for this factor. We show that the p48 isoform of ITAF45, but not the p42 isoform, is crucial for efficient EMCV/Mengovirus replication and for propagation of replicons in human cell culture. Loss of ITAF45 markedly diminishes EMCV and FMDV IRES activities, which can be rescued by re-expression of ITAF45-p48. Interestingly, cell-free translation assays reveal that EMCV IRES activity is less ITAF45-dependent in vitro, in contrast to FMDV, raising questions about the versatile functions of ITAFs in IRES-driven translation. These findings reveal an isoform-specific function of ITAF45 in supporting cardiovirus infection and provide new insights into the complex regulation of IRES-driven translation, with implications for developing targeted antiviral strategies.

molecular biology↗

bioRxiv2024

Eukaryotic initiation factors eIF4F and eIF4B promote translation termination upon closed-loop formation

Eukaryotic translation initiation factor eIF4F, comprising subunits eIF4G, eIF4E, and eIF4A, plays a pivotal role in the 48S preinitiation complex assembly and ribosomal scanning. Additionally, eIF4B enhances the helicase activity of eIF4A. eIF4F also interacts with PABP bound to the poly(A) tail of mRNA, thereby forming a closed-loop structure. PABP, in turn, interacts with eRF3, stimulating translation termination. Here, we employed a reconstituted mammalian system to directly demonstrate that eIF4F potently enhances translation termination. Specifically, eIF4A and eIF4B promote the loading of eRF1 into the A site of the ribosome, while eIF4G1 stimulates the GTPase activity of eRF3 and facilitates the dissociation of release factors following peptide release. We also identified MIF4G as the minimal domain required for this activity and show that eIF4G2/DAP5 can also promote termination. Our findings provide compelling evidence that the closed-loop mRNA structure facilitates translation termination, with PABP and eIF4F directly involved in this process.

molecular biology↗

bioRxiv2024

Transcriptomic Hallmarks of Mortality Reveal Universal and Specific Mechanisms of Aging, Chronic Disease, and Rejuvenation

Health is strongly affected by aging and lifespan-modulating interventions, but the molecular mechanisms of mortality regulation remain unclear. Here, we conducted an RNA-seq analysis of mice subjected to 20 compound treatments in the Interventions Testing Program (ITP). By integrating it with the data from over 4,000 rodent tissues representing aging and responses to genetic, pharmacological, and dietary interventions with established survival data, we developed robust multi-tissue transcriptomic biomarkers of mortality, capable of quantifying aging and change in lifespan in both short-lived and long-lived models. These tools were further extended to single-cell and human data, demonstrating common mechanisms of molecular aging across cell types and species. Via a network analysis, we identified and annotated 26 co-regulated modules of aging and longevity across tissues, and developed interpretable module-specific clocks that capture aging- and mortality-associated phenotypes of functional components, including, among others, inflammatory response, mitochondrial function, lipid metabolism, and extracellular matrix organization. These tools captured and characterized acceleration of biological age induced by progeria models and chronic diseases in rodents and humans. They also revealed rejuvenation induced by heterochronic parabiosis, early embryogenesis, and cellular reprogramming, highlighting universal signatures of mortality, shared across models of rejuvenation and age-related disease. They included Cdkn1a and Lgals3, whose human plasma levels further demonstrated a strong association with all-cause mortality, disease incidence and risk factors, such as obesity and hypertension. Overall, this study uncovers molecular hallmarks of mammalian mortality shared across organs, cell types, species and models of disease and rejuvenation, exposing fundamental mechanisms of aging and longevity.

systems biology↗

bioRxiv2024

Major role of S-glycoprotein in providing immunogenicity and protective immunity in mRNA lipid nanoparticle vaccines based on SARS-CoV-2 structural proteins

Recently we have developed an mRNA lipid nanoparticle (mRNA-LNP) platform providing efficient long-term expression of an encoded gene in vivo after both intramuscular and intravenous application. Based on this platform, we have generated mRNA-LNP coding SARS-CoV-2 structural proteins M, N, S from different virus variants and studied their immunogenicity separately or in combinations in vivo. As a result, all candidate vaccine compositions coding S and N proteins induced excellent anti-RBD and N titers of binding antibodies. T cell responses mainly represented specific CD4+ T cell lymphocyte producing IL-2 and TNF-. mRNA-LNP coding M protein did not show high immunogenicity. High neutralizing activity was detected in sera of mice vaccinated with mRNA-LNP coding S protein (alone or in combinations) against closely related strains but was not detectable or significantly lower against an evolutionarily distant variant. Our data showed that the addition of mRNAs encoding S and M antigens to the mRNA-N in the vaccine composition enhanced immunogenicity of mRNA-N inducing more robust immune response to the N protein. Based on our results, we suggested that the S protein plays a key role in enhancement of immune response to the N protein in the mRNA-LNP vaccine.

immunology↗

bioRxiv2023

Trivalent mRNA vaccine-candidate against seasonal flu with cross-specific humoral immune response

ABSTRACTSeasonal influenza remains a serious global health problem, leading to high mortality rates among the elderly and individuals with comorbidities. It also imposes a substantial economic burden through increased absenteeism during periods of active pathogen circulation. Vaccination is generally accepted as the most effective strategy for influenza prevention. As both influenza A and B viruses circulate and cause seasonal epidemics, vaccines need to include multiple antigens derived from different viral subtypes. While current influenza vaccines are effective, they still have limitations, including narrow specificity for certain serological variants, which may result in a mismatch between vaccine antigens and circulating strains. Additionally, the rapid variability of the virus poses challenges in providing extended protection beyond a single season. Therefore, mRNA technology is particularly promising for influenza prevention, as it enables the rapid development of multivalent vaccines and allows for quick updates of their antigenic composition. mRNA vaccines have already proven successful in preventing COVID-19 by eliciting rapid cellular and humoral immune responses. In this study, we present the development of a trivalent mRNA vaccine candidates, evaluate its immunogenicity using the hemagglutination inhibition assay, and assess its efficacy in animals. We demonstrate the higher immunogenicity of the mRNA vaccine candidates compared to the inactivated split influenza vaccine and its enhanced ability to generate a cross-specific humoral immune response. These findings highlight the potential mRNA technology in overcoming current limitations of influenza vaccines and hold promise for ensuring greater efficacy in preventing seasonal influenza outbreaks.

immunology↗

bioRxiv2023

Introduction of Dicistrovirus IRESs into UAS/SV40-polyA constructs results in premature polyadenylation and strong overexpression of the upstream ORF in Drosophila animals.

To evaluate the properties of insect virus internal ribosomal entry sites (IRESs) for protein expression in Drosophila, we have introduced Cricket Paralysis virus (CrPV) and Drosophila C virus (DCV) IRESs into UAS/SV40-polyA vector. We found that introduction of IRESs induce premature polyadenylation, resulting in both truncation of the mRNA, and an increase in mRNA levels of approximately 40-fold. The increase in mRNA levels was accompanied by increased resistance to nonsense-mediated mRNA decay (NMD)-mediated degradation. Our results suggest that premature polyadenylation increases mRNA stability in the SV40 polyadenylation site-containing constructs, suggesting a novel method for robust overexpression of transgenes in Drosophila.

genetics↗

bioRxiv2023

Monitoring integrated stress response in live Drosophila

Cells exhibit stress responses to various environmental changes. Among these responses, the integrated stress response (ISR) plays a pivotal role as a crucial stress signaling pathway. While extensive ISR research has been conducted on cultured cells, our understanding of its implications in multicellular organisms remains limited, largely due to the constraints of current techniques that hinder our ability to track and manipulate the ISR in vivo. To overcome these limitations, we have successfully developed an internal ribosome entry site (IRES)-based fluorescent reporter system. This innovative reporter enables us to label Drosophila cells, within the context of a living organism, that exhibit eIF2 phosphorylation-dependent translational shutoff - a characteristic feature of the ISR and viral infections. Through this methodology, we have unveiled tissue- and cell-specific regulation of stress response in Drosophila flies and have even been able to detect stressed tissues in vivo during virus and bacterial infections. To further validate the specificity of our reporter, we have engineered ISR-null eIF2S50A mutant flies for stress response analysis. Our results shed light on the tremendous potential of this technique for investigating a broad range of developmental, stress, and infection-related experimental conditions. Combining the reporter tool with ISR-null mutants establishes Drosophila as an exceptionally powerful model for studying the ISR in the context of multicellular organisms.

developmental biology↗

bioRxiv2023

Human tissues exhibit diverse composition of translation machinery

While protein synthesis is vital for the majority of cell types of the human body, diversely differentiated cells require specific translation regulation. This suggests specialization of translation machinery across tissues and organs. Using transcriptomic data from GTEx, FANTOM, and Gene Atlas we systematically explored the abundance of transcripts encoding translation factors and aminoacyl-tRNA synthetases (ARSases) in human tissues. We revised a few known and identified several novel translation-related genes exhibiting strict tissue-specific expression. The proteins they encode include eEF1A1, eEF1A2, PABPC1L, PABPC3, eIF1B, eIF4E1B, eIF4ENIF1, and eIF5AL1. Furthermore, our analysis revealed a pervasive tissue-specific relative abundance of translation machinery components (e.g. PABP and eRF3 paralogs, eIF2B subunits, eIF5MPs, and some ARSases), suggesting presumptive variance in the composition of translation initiation, elongation, and termination complexes. These conclusions were largely confirmed by the analysis of proteomic data. Finally, we paid attention to sexual dimorphism in the repertoire of translation factors encoded in sex chromosomes (eIF1A, eIF2{gamma}, and DDX3), and identified testis and brain as organs with the most diverged expression of translation-associated genes.

molecular biology↗

bioRxiv2022

Longevity and rejuvenation effects of cell reprogramming are decoupled from loss of somatic identity

Partial somatic cell reprogramming has been touted as a promising rejuvenation strategy. However, its association with mechanisms of aging and longevity at the molecular level remains unclear. We identified a robust transcriptomic signature of reprogramming in mouse and human cells that revealed co-regulation of genes associated with reprogramming and response to lifespan-extending interventions, including those related to DNA repair and inflammation. We found that age-related gene expression changes were reversed during reprogramming, as confirmed by transcriptomic aging clocks. The longevity and rejuvenation effects induced by reprogramming in the transcriptome were mainly independent of pluripotency gain. Decoupling of these processes allowed predicting interventions mimicking reprogramming-induced rejuvenation (RIR) without affecting somatic cell identity, including an anti-inflammatory compound osthol, ATG5 overexpression, and C6ORF223 knockout. Overall, we revealed specific molecular mechanisms associated with RIR at the gene expression level and developed tools for discovering interventions that support the rejuvenation effect of reprogramming without posing the risk of neoplasia.

systems biology↗

bioRxiv2022

Robust expression of transgenes in Drosophila melanogaster

Drosophila is a classic experimental system used in fundamental and biopharmaceutical research. Manipulating gene expression in the larvae and adult flies can facilitate basic and translational studies. Here we report a method for robust transgene overexpression in Drosophila melanogaster. This approach is compatible with the UAS/Gal4 gene expression system. The improved expression involves a gene expression cassette that prevents transgenic mRNA transcript degradation by the nonsense-mediated RNA decay pathway. The protection is mediated by CrPV and DCV IRES sequences that apparently encode cryptic polyadenylation sites and NMD inhibitors. Stabilization of the transgene mRNA results in a >40-fold increase in its levels.

genetics↗

bioRxiv2022

Single-domain antibody delivery using an mRNA platform protects against lethal doses of botulinum neurotoxin A

Single-domain antibodies (sdAbs, VHHs, or nanobodies) are a promising tool for the treatment of both infectious and somatic diseases. Their small size greatly simplifies any genetic engineering manipulations. Such antibodies have the ability to bind hard-to-reach antigenic epitopes through long parts of the variable chains, the third complementarity-determining regions (CDR3s). VHH fusion with the canonical immunoglobulin Fc fragment allows the Fc-fusion single-domain antibodies (VHH-Fc) to significantly increase their neutralizing activity and serum half-life. Previously we have developed and characterized VHH-Fc specific to botulinum neurotoxin A (BoNT/A), that showed a 1000-fold higher protective activity than monomeric form when challenged with five times the lethal dose (5 LD50) of BoNT/A. During the COVID-19 pandemic, mRNA vaccines based on lipid nanoparticles (LNP) as a delivery system have become an important translational technology that has significantly accelerated the clinical introduction of mRNA platforms. We have developed an mRNA platform that provides long-term expression after both intramuscular and intravenous application. The platform has been extensively characterized using firefly luciferase (Fluc) as a reporter. An intramuscular administration of LNP-mRNA encoding VHH-Fc antibody made it possible to achieve its rapid expression in mice and resulted in 100% protection when challenged with up to 100 LD50 of BoNT/A. The presented approach for the delivery of sdAbs using mRNA technology greatly simplifies drug development for antibody therapy and can be used for emergency prophylaxis.

molecular biology↗

bioRxiv2021

Multi-omic rejuvenation and lifespan extension upon exposure to youthful circulation

Heterochronic parabiosis (HPB) is known for its functional rejuvenation effects across several mouse tissues. However, its impact on the biological age of organisms and their long-term health remains unknown. Here, we performed extended (3-month) HPB, followed by a 2-month detachment period of anastomosed pairs. Old detached mice exhibited improved physiological parameters and lived longer than control isochronic mice. HPB drastically reduced the biological age of blood and liver based on epigenetic analyses across several clock models on two independent platforms; remarkably, this rejuvenation effect persisted even after 2 months of detachment. Transcriptomic and epigenomic profiles of anastomosed mice showed an intermediate phenotype between old and young, suggesting a comprehensive multi-omic rejuvenation effect. In addition, old HPB mice showed transcriptome changes opposite to aging, but akin to several lifespan-extending interventions. Altogether, we reveal that long-term HPB can decrease the biological age of mice, in part through long-lasting epigenetic and transcriptome remodeling, culminating in the extension of lifespan and healthspan.

cell biology↗

bioRxiv2021

Systematic identification of yeast mutants with increased rates of cell death reveals rapid stochastic necrosis associated with cell division

Cell death plays a major role in development, pathology and aging and can be triggered by various types of acute stimuli which arrest cell growth. However, little is known about chronic cell death in the context of continuing cell division. Here, we performed a genome-wide search for mutants with this type of death in dividing bakers yeast by assaying staining with phloxine B, which accumulates in dead cells. This screen yielded 83 essential and 43 non-essential gene mutants. Three contrasting types of spatial distribution of dead cells in colonies were observed, which corresponded to gene ontology enrichment for (i) DNA replication and repair, RNA processing, chromatin organization, and nuclear transport; (ii) mitosis and cytokinesis; and (iii) vesicular transport and glycosylation/cell wall homeostasis. To study dynamics of cell death in these mutants, we developed methods for analyzing the death of newborn cells (DON) and cell death in real time using microfluidics-based microscopy. These revealed rapid stochastic necrosis during bud generation or cytokinesis without prior division arrest. Increased death during division was associated with common sensitivity to plasma membrane and cell-wall perturbing agents, and could be mitigated by neutral pH stabilization of the medium. This suggests a common downstream type of cell death caused by a wide range of genetic perturbations.

cell biology↗

bioRxiv2021

A standard knockout procedure alters expression of adjacent loci at the translational level

The S. cerevisiae gene deletion collection is widely used for functional gene annotation and genetic interaction analyses. However, the standard G418-resistance cassette used to produce knockout mutants delivers strong regulatory elements into the target genetic loci. To date, its side effects on the expression of neighboring genes have never been systematically assessed. Here, using ribosome profiling data, RT-qPCR, and reporter expression, we investigated perturbations induced by the KanMX module. Our analysis revealed significant alterations in the transcription efficiency of neighboring genes and, more importantly, severe impairment of their mRNA translation, leading to changes in protein abundance. In the "head-to-head" orientation of the neighbor and the deleted gene, knockout often led to a shift of the transcription start site of the neighboring gene, introducing new uAUG codon(s) into the expanded 5 untranslated region (5 UTR). In the "tail-to-tail" arrangement, knockout led to activation of alternative polyadenylation signals in the neighboring gene, thus altering its 3 UTR. These events may explain the so-called neighboring gene effect (NGE), i.e. false genetic interactions of the deleted genes. We estimate that in as much as [~]1/5 of knockout strains the expression of neighboring genes may be substantially (>2-fold) deregulated at the level of translation.

molecular biology↗

bioRxiv2020

Multi-faceted deregulation of gene expression and protein synthesis with age

Protein synthesis represents a major metabolic activity of the cell. However, how it is affected by aging and how this in turn impacts cell function remains largely unexplored. To address this question, herein we characterized age-related changes in both the transcriptome and translatome of mouse tissues over the entire lifespan. Expression of the majority of differentially expressed genes followed a U-shaped curve with the turning point around 3-months-old. We showed that transcriptome changes govern changes in the translatome and are associated with altered expression of genes involved in inflammation, extracellular matrix and lipid metabolism. We also identified genes that may serve as candidate biomarkers of aging. At the translational level, we uncovered sustained down-regulation of a set of 5 terminal oligopyrimidine (5TOP) transcripts encoding protein synthesis and ribosome biogenesis machinery and regulated by the mTOR pathway. For many of them, ribosome occupancy dropped 3-fold or even more. Moreover, with age, ribosome coverage gradually decreased in the vicinity of start codons and increased near stop codons, revealing complex age-related changes in the translation process. Taken together, our results reveal systematic and multi-dimensional deregulation in protein synthesis, showing how this major cellular process declines with age.

systems biology↗