Biology subjects

Mishra, P. P.

Publications and source records attributed to Mishra, P. P..

11 recordsLinked to original sources

bioRxiv2026

Cytoplasmic recapping commences the translation of a micropeptide, MARS from MALAT1 after the removal of an inhibitory RNA G-quadruplex

Long non-coding RNAs (lncRNAs), conventionally defined as RNA transcripts longer than 200 nucleotides that lack protein-coding potential, have emerged as key regulators of diverse biological processes, including metabolism, development, cellular differentiation, and stress responses. They modulate cellular functions through multiple mechanisms, including chromatin remodeling, transcriptional regulation, and the control of RNA stability and translation. Most lncRNAs are transcribed by RNA polymerase II and undergo canonical RNA processing, including splicing, polyadenylation, and 5' N7-methylguanosine (mG) capping. However, whether cytoplasmic recapping of lncRNAs can promote the production of functional micropeptides remains poorly understood. In this study, we investigated the functional consequences of cytoplasmic recapping of MALAT1, a highly abundant and broadly expressed lncRNA. We demonstrate that cytoplasmic processing and recapping of a MALAT1 decay intermediate support the production of a previously uncharacterized micropeptide, designated MARS (MALAT1 Responsive to Stress). We further identify RNA G-quadruplex (rG4) structures in the 5' region of MALAT1 that inhibit MARS translation, revealing an additional layer of structural regulation. MARS expression increases in response to arsenite-induced oxidative stress and is associated with nuclear factor erythroid 2-related factor 2 (Nrf2)-containing complexes. Furthermore, MARS is implicated in the regulation of antioxidant response element-associated genes, including SOD and CAT, suggesting a potential role in cellular antioxidant defense. Collectively, our findings identify a previously unrecognized functional output of cytoplasmic RNA recapping and expand the potential coding repertoire of lncRNAs. This study highlights a mechanism through which cytoplasmic RNA processing may contribute to micropeptide production and suggests a role for MALAT1-derived MARS in oxidative stress-responsive gene regulation.

molecular biology↗

bioRxiv2026

Traditional disease risk factors outperform epigenetic clocks as predictors of non-communicable disease morbidity in a middle-aged cohort

DNA methylation-based epigenetic clocks have been highlighted as promising biomarkers of ageing, and they have been shown to robustly predict morbidity and mortality. However, current literature is lacking a formal analysis of the increased prediction accuracy, or the added value, of the epigenetic clocks over traditional risk factors of common chronic diseases. Here, we have compared the most commonly used epigenetic clocks and traditional risk factors as predictors of incidence of ageing-associated non-communicable chronic disease in a 7-to-9-year follow-up in a middle-aged population cohort (n=1108, aged 34 to 49 years at baseline). In our cohort, a statistical model consisting of a combination of traditional risk factors outperforms any model including an epigenetic clock. The added value of epigenetic clock measurements over simple and affordable traditional risk factors should be clearly established, if epigenetic clocks are to be used in clinical settings or as tools of personal health monitoring.

molecular biology↗

bioRxiv2025

Suppression of non-canonical autophagy induces endothelial and cardiac dysfunction

BackgroundWhile roles for canonical autophagy in the pathophysiology of cardiovascular disease have been established, we have limited understanding of the non-canonical functions of autophagy proteins in this context. LC3-asssociated endocytosis (LANDO) is a novel non-canonical function of autophagy proteins, in which LC3 (microtubule-associated protein light chain 3) is conjugated to early endosome membranes using a portion of the canonical autophagy machinery, and functions in the endocytic recycling of several plasma membrane proteins. Here we ask whether perturbation of LANDO can promote cardiovascular pathogenesis. MethodsCardiac and endothelial functions were assessed by echocardiography and flow-mediated dilatation in mice lacking Rubicon (Rubcn-/-) or the WD domain of ATG16L1 (Atg16l1{Delta}WDki), two known effectors of LANDO. Mice with conditional depletion of Rubicon in the endothelial, myeloid and cardiomyocyte compartments were used as well. Three-dimensional murine cardiac vasculature leakiness was investigated by light sheet fluorescence microcopy. Endothelial activation induced by shear stress was characterized in vitro in primary endothelial cells isolated from murine lungs and human aortic endothelial cells. Associations between genetically predicted expression of candidate genes involved in LANDO and human cardiovascular parameters were studied in the Young Finns Study and the UK Biobank. ResultsCompared to littermate controls, young Rubcn-/- and Atg16l1{Delta}WDki mice showed a decrease in cardiac and endothelial functions, as did mice with endothelium-specific deficiency. VEGFR2 recycling to the plasma membrane and nitric oxide pathway during shear stress were disrupted in LANDO-deficient primary murine and human endothelial cells. Proteomic analysis in primary human aortic endothelial cells revealed an upregulation of intracellular hemoglobin subunit alpha (Hb-) upon shear stress, which was blunted when RUBCN was ablated. Genetic expression studies uncovered several candidate genes related to LANDO that correlated with cardiovascular parameters. These included the retromer complex subunit VPS29, disruption of which decreased Hb- expression levels in human endothelial cells. ConclusionsOur data support a pivotal role of non-canonical functions of autophagy proteins in recycling VEGFR2 upon shear stress activation in endothelial cells together with Hb- expression that may contribute to the etiology of cardiovascular diseases.

physiology↗

bioRxiv2025

Distant Site Mutations in Clinical TEM Beta-Lactamase Variants Enhance Non-Covalent Binding to Ceftazidime: Insights from Spectroscopic and Biophysical Investigations.

{beta}-lactamases retain the central armamentarium against the {beta}-lactams, resulting in surge of antibiotic resistance, primarily due to the hydrolysis of the amide bond of the four-membered ring. This study aimed to investigate the binding interactions of ceftazidime (CAZ) to TEM {beta}- lactamase variants with distant site mutations isolated from clinical setting to explore the cause of their selection and dissemination due to indiscriminate use of {beta}-lactams. Absorbance and fluorescence spectroscopy along with biophysical experimentation indicated facilitated binding of CAZ to the active site of the mutants than the wild type. The CAZ-TEM {beta}-lactamase mutant interactions were predominantly hydrophobic compared to H-bonding and van der Waals forces in the CAZ-wild type complex. Additionally structural alteration to justify more rigid binding of CAZ to the mutants in contrast to the wild type enzyme was established in silico. Therefore, acquisition of distant site mutations with respect to the active site of the {beta}-lactamase variants that rendered conformational flexibility to accommodate CAZ was evidenced. This study provided an insight to the bioactive interaction of CAZ with TEM {beta}-lactamase variants that probably facilitated their selection from clinical settings in response to rampant usage of {beta}-lactams.

microbiology↗

bioRxiv2023

Unveiling the Holliday junction conformational dynamics upon binding with architectural proteins IHF

Integration host factor (IHF) of E. coli is a nucleoid-associated protein with diverse roles in DNA packaging, viral DNA integration, and recombination. IHF binds to duplex DNA containing a 13 bp consensus sequence with nanomolar affinity and induces a significant bend of approximately 160{degrees} upon binding. While the Wild type IHF (WtIHF) is involved in DNA bending, thereby facilitating the integration of foreign DNA into the host genome, its engineered version, Single chain IHF (ScIHF) was designed for specific genetic engineering and biotechnological applications. We investigated interaction of the two IHF variants with Holliday junctions (HJ), crucial intermediates in DNA repair and homologous recombination. Our finding demonstrate that both variant of IHF binds to HJs with high affinity in presence of the consensus sequence, indicating a structure-based recognition mechanism. HJs are dynamic structures that can adopt open or stacked conformation. The open conformation facilitates processes like branch migration and strand exchange. Through quantitative binding studies using microscale thermophoresis, we determined the binding of IHF to four-way DNA junctions that harboured two specific binding sequences H & H1. Circular dichroism (CD) experiments revealed the proteins impact on the junction conformation. This was further confirmed by Single molecule Forster resonance energy transfer (smFRET) technique that was used to examine the binding of IHF to the junction and its effect on the dynamicity of junction conformation. We also probed the population distribution of junction conformations. Interestingly, our results revealed that binding of both WtIHF & ScIHF shifts the population towards the open conformation of the junction and stabilised it in that conformation. In summary, our findings demonstrate that IHF binds HJs with a strong affinity and has a stabilizing effect on maintaining the junctions open conformation.

biophysics↗

bioRxiv2022

Methylation status of VTRNA2-1/nc886 is stable across human populations, monozygotic twin pairs and in majority of somatic tissues

Aims and methodsOur aim was to characterise the methylation level of a polymorphically imprinted gene, VTRNA2-1/nc886, in human populations and somatic tissues. We utilised 48 datasets, consisting of >30 different tissues and >30 000 individuals. ResultsWe show that the nc886 methylation status is associated with twin status and ethnic background, but the variation between populations is limited. Monozygotic twin pairs present concordant methylation, while [~]30% of dizygotic twin pairs present discordant methylation in the nc886 locus. The methylation levels of nc886 are uniform across somatic tissues, except in cerebellum and skeletal muscle. ConclusionWe hypothesize that the nc886 imprint is established in the oocyte and that after implantation, the methylation status is stable, excluding a few specific tissues.

molecular biology↗

bioRxiv2021

Probing the molecular mechanism of action of Deinococcus radiodurans RecD2 at single-molecule resolution

Helicases are ATP-driven molecular machines that directionally remodel nucleic acid polymers in all three domains of life. Helicases are responsible for resolving double-stranded DNA (dsDNA) into separate single-strands and this activity is essential for DNA replication, nucleotide excision repair, and homologous recombination. RecD2 from Deinococcus radiodurans (DrRecD2) has important contributions towards the organisms unusually high tolerance to gamma radiation and hydrogen peroxide. Although previous X-ray Crystallography studies have revealed the structural characteristics of the protein, the direct experimental evidence regarding the dynamics of the DNA unwinding process by DrRecD2 in the context of other accessory proteins is yet to be found. In this study, we have probed the exact binding event and processivity of DrRecD2 at single-molecule resolution using Protein-induced fluorescence enhancement (smPIFE) and Forster resonance energy transfer (smFRET). We have found that the protein prefers to bind at the 5 terminal end of the single-stranded DNA (ssDNA) by Drift and has helicase activity even in absence of ATP. However, a faster and iterative mode of DNA unwinding was evident in presence of ATP. The rate of translocation of the protein was found to be slower on dsDNA compared to ssDNA. We also showed that DrRecD2 is recruited at the binding site by the single-strand binding protein (SSB) and during the unwinding, it can displace RecA from ssDNA.

biophysics↗

bioRxiv2021

Single Molecule Analysis of differential functional mechanisms of MtRecG while being exposed to variants of stalled replication-fork

Helicases are motor proteins involved in multiple activities to carry out manipulation of the nucleic acids for efficient gene regulation. In case of roadblocks that can lead the replication machinery to get halted, a complex molecular surveillance system utilizing helicases as its key player ensures the halted fork to resume its duplication process. RecG, belonging to the category of Superfamily-2 plays a vital role in rescuing different kinds of stalled fork. Here, through adoption of single-molecule techniques we have attempted to probe the DNA unwinding features by RecG and tried to capture several stages of genetic rearrangement. An elevated processivity of RecG has been observed for the kinds of stalled fork where progression of lagging daughter strand is ahead than that of the leading strand. Through precise alteration of its function in terms of unwinding, depending upon the substrate DNA, RecG catalyzes the formation of Holliday junction from a stalled fork DNA. In summary, we have featured that RecG adopts asymmetric mode of locomotion to unwind the lagging daughter strand to facilitate Holliday junction creation which acts as a suitable intermediate for recombinational repair pathway.

biophysics↗

bioRxiv2020

Vital Insights into Prokaryotic Genome Compaction by Nucleoid-Associated Protein (NAP) and Illustration of DNA Flexure Angles at Single Molecule Resolution

Integration Host Factor (IHF) is a heterodimeric site-specific nucleoid-associated protein (NAP) well known for its DNA bending ability. The binding is mediated through the narrow minor grooves of the consensus sequence, involving van der-Waals interaction and hydrogen bonding. Although the DNA bend state of IHF has been captured by both X-ray Crystallography and Atomic Force Microscopy (AFM), the range of flexibility and degree of heterogeneity in terms of quantitative analysis of the nucleoprotein complex has largely remained unexplored. Here we have monitored and compared the trajectories of the conformational dynamics of a dsDNA upon binding of wild-type (wt) and single-chain (sc) IHF at millisecond resolution through single-molecule FRET (smFRET). Our findings reveal that the nucleoprotein complex exists in a Slacked-Dynamic state throughout the observation window where many of them have switched between multiple Wobbling States in the course of attainment of packaged form. A range of DNA Flexure Angles has been calculated that give us vital insights regarding the nucleoid organization and transcriptional regulation in prokaryotes. This study opens up an opportunity to improve the understanding of the functions of other nucleoid-associated proteins (NAPs) by complementing the previous detailed atomic-level structural analysis, which eventually will allow accessibility towards a better hypothesis.

biophysics↗

bioRxiv2020

DNA methylation signatures of aggression and closely related constructs: A meta-analysis of epigenome-wide studies across the lifespan

DNA methylation profiles of aggressive behavior may capture lifetime cumulative effects of genetic, stochastic, and environmental influences associated with aggression. Here, we report the first large meta-analysis of epigenome-wide association studies (EWAS) of aggressive behavior (N=15,324 participants). In peripheral blood samples of 14,434 participants from 18 cohorts with mean ages ranging from 7 to 68 years, 13 methylation sites were significantly associated with aggression (alpha=1.2x10-7; Bonferroni correction). In cord blood samples of 2,425 children from five cohorts with aggression assessed at mean ages ranging from 4 to 7 years, 83% of these sites showed the same direction of association with childhood aggression (r=0.74, p=0.006) but no epigenome-wide significant sites were found. Top-sites (48 at a false discovery rate of 5% in the peripherl blood meta-analysis or in a combined meta-analysis of peripheral blood and cord blood) have been associated with chemical exposures, smoking, cognition, metabolic traits, and genetic variation (mQTLs). Three genes whose expression levels were associated with top-sites were previously linked to schizophrenia and general risk tolerance. At six CpGs, DNA methylation variation in blood mirrors variation in the brain. On average 44% (range=3-82%) of the aggression-methylation association was explained by current and former smoking and BMI. These findings point at loci that are sensitive to chemical exposures with potential implications for neuronal functions. We hope these results to be a starting point for studies leading to applications as peripheral biomarkers and to reveal causal relationships with aggression and related traits.

genetics↗

bioRxiv2020

Genome-wide association studies identify 137 loci for DNA methylation biomarkers of ageing

Biological ageing estimators derived from DNA methylation (DNAm) data are heritable and correlate with morbidity and mortality. Leveraging DNAm and SNP data from >41,000 individuals, we identify 137 genome-wide significant loci (113 novel) from meta-analyses of four epigenetic clocks and epigenetic surrogate markers for granulocyte proportions and plasminogen activator inhibitor 1 levels, respectively. We report strong genetic correlations with longevity and lifestyle factors such as smoking, education, and obesity. Significant associations are observed in polygenic risk score analysis and to a lesser extent in Mendelian randomization analyses. This study illuminates the genetic architecture underlying epigenetic ageing and its shared genetic contributions with lifestyle factors and longevity.

genetics↗