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Results for “molecular biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2019

Enhancer-promoter association determines Sox2 transcription regulation in mouse pluripotent cells

Distal enhancer-promoter associations are essential for transcription control and emerging as a common epigenetic way to determine gene expression in eukaryotes. However, it remains as an uncultivated land on how their diversity influences gene transcription during development. In this study, we select three defined Sox2 associated enhancers (E1, E2 and E3), representing different distal interaction categories, to further explore their biological effects. We construct three enhancer-knockout cells via CRISPR/Cas9 system in mouse embryonic stem cells. Results show that these associations carry out various biological features. Embryonic stem cell specific association (E1) speeds up cell cycle and involves in cardiac development, which has been validated in vivo. In contrast, the indirect one (E2) restrains nerve differentiation and has potential effect on lipid metabolism. The common one (E3) promotes nerve differentiation and inhibits oxidation-reduction process. Together, these different associations determine Sox2 transcription and function specificity in mouse embryonic stem cells. Our study will enable a way for exploring miscellaneous spatiotemporal gene transcription control and advancing quantitative knowledge by utilizing three-dimensional genomic information.\n\nSummary statementSox2 transcription is complicated in mouse embryonic stem cells. It involves in heavily enhancer-promoter associations, which might make it a suitable model for phase-separation study. To decompose this cooperative regulation, diverse interactions are investigated. The biological effects have been explored in multiple perspectives. This may represent a quantitative way to explore and a potential new strategy to transcription control study.

molecular biology↗

bioRxiv2026

Tumor Suppression across the Human miRNome Associates with Guanine-Rich Precursor Terminal Loops

MicroRNAs (miRNAs) play essential regulatory roles in controlling cell growth, proliferation, and differentiation in cancer. While functional studies have identified numerous oncogenic (oncomiRs) and tumor suppressor (TS) miRNAs, the structural features that differentiate these groups remain poorly understood. Here, we performed a comprehensive sequence analysis of 955 human pre-miRNA terminal loops (TLs), focusing on enrichment of single guanine (G) and GG dinucleotides. A quantitative G enrichment score was used to define 42 G-rich TL miRNAs and 17 G-free TL miRNAs as controls. Functional roles of these miRNAs were curated from 757 publications. The results show that G-rich TL miRNAs consistently display higher TS/oncomiR ratios than G-free TL miRNAs across most cancer types, with a significant enrichment observed in lung cancer (p = 0.023). Focusing on miR-139, a TS miRNA with a G-rich TL, integrative analysis of publicly available transcriptomic and proteomic data revealed its consistent downregulation across all stages of lung adenocarcinoma (LUAD), accompanied by reciprocal overexpression of its validated oncogenic target, CCNB1. These findings highlight the biological relevance of G-rich TL structures in miRNA-mediated tumor suppression in lung cancer, suggesting their consideration in future therapeutic strategies aimed at restoring vulnerable TS miRNAs.

molecular biology↗

bioRxiv2026

Expanded Proteome Coverage Powered by Advanced Ion Processing Enables Deep Single-Cell Drug Response Subtyping in Human Stem Cell Derived Cardiomyocytes

Single-cell proteomics (SCP) enables the study of cellular heterogeneity at the functional level but remains limited by incomplete proteome coverage and high data missingness. Here, we present an enhanced label-free SCP workflow that leverages the timsUltra AIP mass spectrometry platform equipped with the Athena Ion Processor (AIP). Across a controlled dilution series of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), AIP-enabled acquisition consistently increased proteome depth and detection consistency across cells at all input levels. In single iPSC-CMs, the timsUltra AIP quantified up to 3,858 protein groups, averaging [~]1,300 proteins per cell, enabling robust proteome-level classification of cardiomyocyte subtypes. Using a reference-based protein classifier, cells were stratified into mature cardiomyocytes and less differentiated cell states, revealing substantial baseline heterogeneity. Importantly, increased single-cell sensitivity translated directly into biological insight, as approximately 30% of differentially expressed proteins associated with subtype-specific drug responses were detected exclusively by timsUltra AIP. Application of this workflow to PR-364 (a mitophagy boosting drug) dose-response experiment uncovered distinct, subtype-dependent pathway adaptations. Mature cardiomyocytes exhibited dose-dependent increases in mitochondrial and metabolic pathway activity, while immature cells showed enrichment of cytoskeletal and developmental programs. These effects were partially obscured in simulated bulk analyses, highlighting the value of single-cell resolution. Together, these results demonstrate that improved fragment ion transmission and utilization translate directly into enhanced biological insight, enabling more comprehensive and functionally relevant single-cell proteomics.

molecular biology↗

bioRxiv2026

Nitrogen-Responsive Extracellular Proteomics Reveals Evidence for a Novel Heterocyst-Specific Protein Secretion Pathway in Anabaena

Nitrogen availability is a major factor governing the physiology, ecology, and metabolism of cyanobacteria. Here, we performed a comparative extracellular proteomic analysis of Anabaena sp. PCC 7120 grown under nitrate-replete and diazotrophic conditions. Using LC-MS/MS, we identified 115 extracellular proteins in nitrate-grown cultures and 113 proteins under N2-fixing conditions. Remarkably, SignalP 6.0 predicted canonical signal peptides in only [~]22% of the identified proteins, suggesting that extracellular protein export in Anabaena predominantly occurs through non-classical secretion mechanisms, potentially involving extracellular vesicles or other unrecognized pathways. Six highly abundant extracellular proteins (Alr2938, Alr4550, Alr2328, All4121, Alr0528, and Alr0529) were detected under both nitrogen regimes. In contrast, All4337, Alr0608, and All3093 were preferentially enriched under nitrate-replete conditions, whereas Alr0267 and Alr1050 emerged among the most abundant extracellular proteins during diazotrophic growth. Notably, an Alr0267-GFP fusion protein was detected exclusively in heterocysts, the specialized N2-fixing cells of Anabaena, with GFP fluorescence concentrated at the cell periphery. The extracellular localization of Alr0267 is particularly intriguing because heterocysts are surrounded by specialized polysaccharide and glycolipid envelope layers that establish the microoxic environment required for nitrogenase activity. The apparent export of Alr0267 across these barriers provides evidence for a previously unrecognized heterocyst-associated protein secretion pathway. Together, these findings reveal a nitrogen-responsive extracellular proteome and provide the first evidence for heterocyst-specific extracellular protein secretion. This work advances our understanding of heterocyst biology and protein trafficking while laying a foundation for engineering Anabaena as a sustainable photosynthetic platform for secreting high-value proteins using sunlight, CO2, N2, and mineralized water.

molecular biology↗

bioRxiv2026

Adeno-Associated Virus (AAV) Synthetic Inverted Terminal Repeats Enhance Tissue-Specific Transduction and Alter the Vector Induced Stress Response

While adeno-associated virus (AAV) vectors have shown therapeutic benefit in clinical applications, noted challenges include low transduction efficiencies, poor cellular targeting, and vector related adverse events. Recently, it was demonstrated that a rationally designed synthetic inverted terminal repeat (SynITR) altered the AAV vector-induced DNA damage response and abrogated apoptosis in human embryonic stem cells. To explore the utility of AAV-SynITR for diverse gene therapy applications, vector production, transduction, and the cellular response were evaluated in various contexts. Regarding production, SynITR preparations exhibited comparable titers to wtITR in a serotype/transgene-independent manner. Despite slightly decreased transduction efficiency in various cell lines, intravenous administration of AAV8 vectors showed SynITR enhanced transduction in a tissue-specific manner in liver (>7-fold) and kidney and pancreas (>2-fold) at equivalent vector copy numbers; however, no differences were observed in muscle/heart/spleen tissues. Interestingly, persistent {gamma}H2AX, a marker of aging/chronic inflammation, was abundant in the liver and spleen following wtITR (but not SynITR) transduction. In human corneas, SynITR enhanced transduction up to 16-fold over wtITRs. These data demonstrate that SynITRs elicit tissue-specific transduction enhancement and alter the cellular stress response. Importantly, the SynITRs offer an alternative context to elucidate wtITR biology for targeted, enhanced, and potentially safer human gene therapy.

molecular biology↗

bioRxiv2026

A multi-scale structural and biophysical atlas of TCR-peptide-HLA recognition dynamics

Dynamic interactions between T cell receptor (TCR) and peptide-human leukocyte antigen (pHLA) complexes are central to peptide-specific immune recognition, influencing T cell activation and immune responses. While structural biology has provided valuable static structures of TCR-pHLA complexes, systematic datasets capturing their dynamic and interaction patterns remain limited. Here, we present DynaTPH, a curated structural dynamics dataset of human TCR-pHLA complexes. DynaTPH integrates TCR-pHLA structures, covering both HLA class I and class II complexes, and extends these static structural resources with standardized molecular dynamics simulations and derived biophysical properties. Through a multi-stage filtering procedure, we identified 256 representative complexes and performed standardized all-atom molecular dynamics simulations for each system, corresponding to a cumulative simulation time of 38.4 s. The dataset includes static structures, trajectories, corresponding frames, and derived physicochemical properties, including hydrogen bonds, intermolecular contacts, solvent accessibility, and backbone flexibility. By capturing the conformational flexibility and dynamic interaction patterns across diverse TCR-pHLA interfaces, DynaTPH extends static structural resources with multidimensional biophysical information. This dataset enables systematic investigation of TCR-pHLA recognition dynamics and supports applications in TCR engineering, vaccine design, and immune tolerance research and artificial intelligence-driven computational immunology.

molecular biology↗

bioRxiv2017

The small non-coding vault RNA1-1 acts as a riboregulator of autophagy

Vault RNAs (vtRNA) are small, 88-100nt non-coding RNAs found in many eukaryotes. Although they have been linked to drug resistance, apoptosis and nuclear transport, their function remains unclear. Here we show that a human vtRNA, RNA1-1, specifically binds to the autophagy receptor sequestosome-1/p62. Antisense-mediated depletion of vault RNA1-1 augments, whereas increased vault RNA1-1 expression restricts, autophagic flux in a p62-dependent manner. Bulk autophagy induced by starvation reduces the levels of vault RNA1-1 and the fraction of RNA-bound p62. These findings show that RNAs can act as riboregulators of biological processes by interacting with proteins, and assign a function to a vault RNA.

molecular biology↗

bioRxiv2020

Post-transcriptional modulation of the SigF regulon in Mycobacterium smegmatis by the PhoH2 toxin-antitoxin.

PhoH2 proteins are highly conserved across bacteria and archaea yet their biological function is poorly characterised. We examined the growth profiles of Mycobacterium smegmatis strains mc2155 and mc2155 {Delta}phoH2 and observed the same growth profile and growth rate in a variety of conditions. In light of the comparable growth rates, we used RNAseq to provide a snapshot of the differences between the transcriptomes of M. smegmatis mc2155 and M. smegmatis mc2155 {Delta}phoH2 during normal growth. At 48 hours, elevated expression of the sigF regulon and its predicted regulatory cascade was observed in{Delta} phoH2 relative to wild type. In biochemical assays, PhoH2 showed specific activity toward sigF mRNA insinuating a role of PhoH2 in modulating the pool of sigF mRNA in the cell during normal growth, adding further complexity to the repertoire of reported mechanisms of post-translational regulation. Multiple copies of the preferred target site of PhoH2 were identified in loops of the sigF mRNA structure, leading us to propose a mechanism for the activity of PhoH2 that is initiated after assembly on specific single-stranded loops of RNA. We hypothesise that PhoH2 is a toxin-antitoxin that contributes to the regulation of SigF at a post-transcriptional level through targeted activity on sigF mRNA. This work presents the first evidence for post-transcriptional regulation of SigF along with the biological function of PhoH2 from M. smegmatis. This also has implications for the highly conserved PhoH2 toxin-antitoxin module across the mycobacteria including the important human pathogen M. tuberculosis.

molecular biology↗

bioRxiv2020

Ethylenediaminetetraacetic acid promotes the accumulation of nitric oxide

Ethylenediaminetetraacetic acid (EDTA) is a well-established chelating agent used in industry, agriculture, food, and medicine. However, the analysis of an EDTA-sensitive Arabidopsis thaliana mutant revealed that EDTA can significantly promote nitric oxide (NO) accumulation, indicating that EDTA has unexpected biological functions beyond its chelating activity. This finding challenges our current understanding about the effects of EDTA on biological systems. One Sentence SummaryAn analysis of ethylenediaminetetraacetic acid (EDTA)-sensitive mutants suggests that EDTA can promote the accumulation of nitric oxide.

molecular biology↗

bioRxiv2021

Determination of m6A frequency utilizing 4SedTTP-RT Ligation Assisted PCR (SLAP) in viral and cellular long non-coding RNAs

N6-methyladenosine is one of the most abundant epitranscriptomic signatures that can affect every aspect of RNA biology, from structure and stability to intra- and intermolecular interactions. The accurate quantitative assessment of RNA stoichiometry at single-nucleotide resolution is a prerequisite to evaluate the biological significance of m6A in the context of specific RNA. We have developed a new method, termed 4-Selenothymidine 5-triphosphate reverse transcription and Ligation Assisted PCR analysis (SLAP), for quantitative and unbiased assessment of the m6A fraction on target RNA. The inclusion of thymidine triphosphate derivative during reverse transcription discourages base pair formation with m6A resulting in the reactions cessation, while maintaining normal A-T base pairing. The site-specific ligation of the resulting cDNAs with adapters, followed by amplification, generates two distinct products that reflect the modified and unmodified fraction of the analyzed RNA. These PCR products are subsequently separated by gel electrophoresis and quantified using densitometric analysis. We applied the SLAP to verify the position and assess the frequency of m6A sites present on two exemplary long non-coding RNAs. We assessed the SLAP specificity, accuracy, and sensitivity, proving the applicability of this method for the m6A analysis on less abundant transcripts. Overall, this method constitutes an extension of the birds-eye view of RNA m6A landscape provided by epitranscriptome-wide analyses by delivering quantitative assessment of modification frequency and can therefore aid the understanding of the consequences of m6A on biological processes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/460679v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@15845aeorg.highwire.dtl.DTLVardef@46a95aorg.highwire.dtl.DTLVardef@118633dorg.highwire.dtl.DTLVardef@1b52f9_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

PlantRNA 2.0 : an updated database dedicated to tRNAs of photosynthetic eukaryotes

PlantRNA (http://plantrna.ibmp.cnrs.fr/) is a comprehensive database of transfer RNA (tRNA) gene sequences retrieved from fully annotated nuclear, plastidial and mitochondrial genomes of photosynthetic organisms. In the first release (PlantRNA 1.0), tRNA genes from 11 organisms were annotated. In this second version, the annotation was implemented to 51 photosynthetic species covering the whole phylogenetic tree of photosynthetic organisms, from the most basal group of Archeplastida, the glaucophyte Cyanophora paradoxa, to various land plants. Transfer RNA genes from lower photosynthetic organisms such as streptophyte algae or lycophytes as well as extremophile photosynthetic species such as Eutrema parvulum were incorporated in the database. As a whole, circa 37 000 tRNA genes were accurately annotated. In the frame of the tRNA genes annotation from the genome of the Rhodophyte Chondrus crispus, non-canonical splicing sites in the D- or T- regions of tRNA molecules were identified and experimentally validated. As for PlantRNA 1.0, comprehensive biological information including 5- and 3-flanking sequences, A and B box sequences, region of transcription initiation and poly(T) transcription termination stretches, tRNA intron sequences and tRNA mitochondrial import are included.

molecular biology↗

bioRxiv2022

Analytic representation of inhomogeneous-resolution maps of three-dimensional scalar fields

Refinement of macromolecular atomic models versus experimental maps in cryo-electron microscopy and crystallography is a critical step in structure solution. For an appropriate comparison, model maps should mimic imperfections of the experimental ones, mainly atomic disorder and its limited resolution, often inhomogeneous over the molecule. We construct these model maps as a sum of atomic contributions expressed through a specially designed function describing a solitary spherical wave. Thanks to this function, atomic contributions analytically depend on both an atomic disorder and the local resolution, a value associated now with each atom. Such fully analytic dependence of inhomogeneous-resolution map values on model parameters permits an efficient refinement of all these parameters together and, beyond structural biology, opens a way to solve similar problems in other research domains. One-Sentence SummaryAn analytic decomposition of 3D-oscillating functions results in efficient tools to calculate maps and refine atomic models.

molecular biology↗

bioRxiv2023

Ribosomal A-site interactions with near-cognate tRNAs drive stop codon readthrough

tRNAs serve as a dictionary for the ribosome translating the genetic message from mRNA into a polypeptide chain. Besides this canonical role, tRNAs are involved in other processes like programmed stop codon readthrough (SC-RT). There, tRNAs with near-cognate anticodons to stop codons must outcompete release factors and incorporate into the ribosomal decoding center to prevent termination and allow translation to continue. However, not all near-cognate tRNAs promote efficient SC-RT. Here, we demonstrate that those that do, establish critical contacts between their anticodon stem (AS) and ribosomal proteins Rps30/eS30 and Rps25/eS25 forming the decoding site. Unexpectedly, the length and well-defined nature of the AS determines the strength of these contacts, which is reflected in organisms with reassigned stop codons. These findings open a new direction in tRNA biology that should facilitate the design of artificial tRNAs with specifically altered decoding abilities.

molecular biology↗

bioRxiv2025

A novel Notch and WNT signaling mechanism contribute to pediatric DCM: a pathway to new therapeutics.

BackgroundTherapies for pediatric idiopathic dilated cardiomyopathy (iDCM) are extrapolated from adult heart failure despite limited efficacy, suggesting fundamental biological differences. Our prior transcriptomic studies indicate activation of developmental signaling pathways, including Notch and WNT, in pediatric iDCM; however, their mechanistic contribution remains unknown. We tested whether reactivation of Notch and WNT/{beta}-catenin signaling drives pathological remodeling in postnatal hearts and whether pathway inhibition improves cardiac function. MethodsWe developed a juvenile rat model to reproduce age-dependent molecular features of pediatric iDCM using {beta}-adrenergic stimulation (isoproterenol, ISO) and secreted frizzled-related protein-1 (sFRP1), a circulating WNT modulator elevated in children with DCM. Cardiac function was assessed by echocardiography; pathway activation by immunoblotting and transcriptomics; myocardial stiffness by atomic force microscopy. Findings were compared with explanted pediatric and adult human myocardium. ResultsExplanted pediatric, but not adult iDCM hearts exhibited increased nuclear and cytoplasmic Notch intracellular domain (NICD) and {beta}-catenin. Combined ISO and sFRP1 treatment recapitulated key features of pediatric disease, including ventricular dilation, reduced ejection fraction, reactivation of the fetal gene program, and increased myocardial stiffness in the absence of fibrosis or hypertrophy. Bulk and single-nucleus RNA sequencing identified cardiomyocyte-specific activation of Notch and WNT pathways and reduced intercellular signaling diversity. Mechanistically, {beta}-catenin silencing attenuated Notch target gene activation and pathological remodeling in vitro. Pharmacologic Notch inhibition reduced NICD and {beta}-catenin accumulation, improved ventricular function, and normalized myocardial stiffness in vivo. ConclusionPediatric iDCM is characterized by pathological co-activation of developmental Notch-WNT signaling pathways that are not observed in adult disease. Reactivation of this axis promotes maladaptive remodeling and myocardial stiffening, and its inhibition improves cardiac function. These findings establish developmental signaling reactivation as a central mechanism of pediatric iDCM and support age-specific therapeutic strategies.

molecular biology↗

bioRxiv2019

A primer genetic toolkit for exploring mitochondrial biology and disease using zebrafish

Mitochondria are a dynamic eukaryotic innovation that play diverse roles in biology and disease. The mitochondrial genome is remarkably conserved in all vertebrates, encoding the same 37 gene set and overall genomic structure ranging from 16,596 base pairs (bp) in the teleost zebrafish (Danio rerio) to 16,569 bp in humans. Mitochondrial disorders are amongst the most prevalent inherited diseases affecting roughly 1 in every 5000 individuals. Currently, few effective treatments exist for those with mitochondrial ailments, representing a major unmet patient need. Mitochondrial dysfunction is also implicated to be a common component of a wide variety of other human illnesses ranging from neurodegenerative disorders like Huntingtons disease and Parkinsons disease to autoimmune illnesses such as multiple sclerosis and rheumatoid arthritis. The electron transport chain (ETC) component of mitochondria is critical for mitochondrial biology and defects can lead to many mitochondrial disease symptoms. Here we present a publicly available collection of genetic mutants created in highly conserved, nuclear-encoded mitochondrial genes in Danio rerio. The zebrafish system represents a potentially powerful new opportunity for the study of mitochondrial biology and disease due to the large number of orthologous genes shared with humans and the many advanced features of this model system from genetics to imaging. This collection includes 22 mutant lines in 18 different genes created by locus-specific gene editing to induce frameshift or splice acceptor mutations leading to predicted protein truncation during translation. Also included are 6 lines created by the random insertion of the gene-breaking transposon (GBT) protein trap cassette. All of these targeted mutant alleles truncate conserved domains of genes critical to the proper function of the ETC or genes that have been implicated in human mitochondrial disease. This collection is designed to accelerate the use of zebrafish to study of many different aspects of mitochondrial function with the goal of widening our understanding of their role in biology and human disease.

molecular biology↗

bioRxiv2026

DNA cut-ligation cyclization surpasses J-Factor limit by order of magnitude

We demonstrate DNA circularization efficiencies more than threefold greater than those predicted by classical Jacobson-Stockmayer theory for the ligation of linear double-stranded DNA. To quantify deviations from classical expectations, we experimentally calibrated the J-factor by ligating 452 bp DNA fragments bearing pre-cut, purified 4-nt overhangs at the optimal shortest minicircle length and at the highest DNA concentration; this reference value then enabled accurate calculation of expected cyclization efficiencies across the full range of DNA lengths (452-952 bp) and the three concentrations examined. These elevated cyclization efficiencies are enabled by simultaneous restriction cutting and ligation using the Type IIS enzyme BsaI-HFv2 in combination with T4 DNA ligase under finely optimized buffer conditions, yielding a 3.4-fold improvement over the classical estimate and achieving 75% cyclization at a high, practically relevant DNA concentration of 120 ng/{micro}l. We also identified a second enzyme, Esp3I, that exceeds the classical expectations by 2.3-fold (though less efficiently than BsaI-HFv2), while BbsI cut-ligation systematically underperformed expectations, providing insight into possible mechanisms underlying the outstanding performance of the first two enzymes. These results reveal a biologically mediated exception that overcomes the long-standing mechanistic expectations of Jacobson- Stockmayer theory and highlight the value of systematically screening enzyme combinations to discover additional systems capable of highly efficient intramolecular ligation.

molecular biology↗

bioRxiv2025

Internally twin-Strep tagged CD63 for rapid and efficient isolation of engineered extracellular vesicles encapsulating functional proteins

Extracellular vesicles (EVs) are important mediators of intercellular communication. Achieving high purity of intact engineered EVs through separation from the overwhelming background of unmodified vesicles and cellular or serum-derived contaminants remains a major challenge, yet is critical for fundamental studies of EV biology and EV-based therapeutics. To address this unmet need, we applied structure-guided engineering to canonical EV tetraspanins. Focusing on CD63 and CD9, we identified sites for internal insertion of the twin-Strep tag via flexible linkers into unstructured regions of their large extracellular loops. This design retains functional properties of the scaffolds while providing a robust molecular handle enabling rapid, efficient, and selective antibody-free affinity-based isolation of intact EVs using advanced Strep technology. Fusing proteins of interest (POIs) to the C-terminus of these scaffolds, either directly or via a photoactivatable protein, allowed POI loading into the lumen of twin-Strep tagged EVs with optional optogenetic control of POI release. Proteomic analysis confirmed the high purity of captured engineered EVs with >96% of contaminants removed, enabling sensitive detection of numerous EV-associated proteins. We also report the efficient removal of contaminating virus particles resembling EVs in size and density. Purified engineered EVs successfully delivered an encapsulated fluorescent reporter into target cells, bypassing dye labeling commonly used to track EVs. We demonstrate endosomal escape of the reporter, facilitated by EV decoration with vesicular stomatitis virus glycoprotein, and cytoplasmic release upon optogenetic activation. Our toolbox may serve as a broadly applicable strategy for the efficient production of intact and highly pure engineered EVs to support potential fundamental or translational EV studies.

molecular biology↗

bioRxiv2020

Nsp1 of SARS-CoV-2 Stimulates Host Translation Termination

The Nsp1 protein of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of SARS-CoV-2 Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1 and the eRF1 and ABCE1 proteins. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex simultaneously with eRF1, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates translation termination in the stop codon recognition stage at all three stop codons. This stimulation targets the release factor 1 (eRF1) and does not affect the release factor 3 (eRF3). The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and removal them from the pool of the active ribosomes.

molecular biology↗