Biology subjects

Mishra, R. K.

Publications and source records attributed to Mishra, R. K..

At least 19 recordsLinked to original sources

bioRxiv2026

Functional dissection of ultraconserved non-coding regions present near the Hoxd complex in vertebrates

Ultraconserved non-coding elements (UCEs) exhibit near-perfect sequence conservation across vertebrate genomes, implying severe evolutionary constraint and essential biological functions. Near the vertebrate Hoxd complex, a master regulator of axial patterning and limb morphogenesis, three Conserved Regions (CR1, CR2, and CR3), each longer than 200 bp, show exceptional sequence conservation and drive robust regulatory activity in transient reporter assays. To investigate their endogenous in vivo functions, we used CRISPR-Cas9 genome editing to generate a series of deletions of varying sizes spanning CR2 and CR3, individually and in combination, within hoxd complex in zebrafish. Screening thousands of founder embryos revealed a clear size and conservation-dependent relationship between deletion extent and viability: complete deletion of core CR2, CR3, or the combined CR2/3 loci could not be transmitted through the germline, whereas the smaller and least-conserved deletion, a 626-bp subregion of CR2, was the only allele to yield a stable, homozygous-viable line. Our results indicate that the core ultraconserved sequences are developmentally indispensable, whereas flanking, less conserved sequences can be removed without consequence. Detailed phenotypic and molecular characterization of the mutant embryos revealed normal primary and secondary body axis formation, intact somite segmentation, and unperturbed craniofacial and pectoral fin development. Expression of hoxd complex genes showed no significant change in mutant embryos by RNA-ISH or RT-qPCR, although RT-qPCR revealed a consistent, modest upward trend at 24 and 48 hpf, raising the possibility that the deleted subregion normally contributes a weak repression of overall hoxd complex genes transcription. While this study reveals some functional aspects of ultraconserved elements, much more remains to be uncovered.

developmental biology↗

bioRxiv2025

Coronin1C SUMOylation modulates filopodia formation, neuritogenesis, and neuronal differentiation

Actin dynamics in the cytosol and at the cell periphery are critical for metabolic processes and the formation of cellular projections. Coronin1C, a versatile cytoskeletal regulator, is an actin-binding protein that associates with actin at the leading edge of a cell. Consequences of Coronin1C interactions on actin dynamics enable actin-mediated filopodia and neurite formation. Here, we report that Coronin1C is SUMOylated at multiple lysine residues in its carboxy-terminus, preferentially by SUMO1. SUMOylation of Coronin1C is critically required for efficient neurite formation and extensive cellular projections during neuronal differentiation. Moreover, SUMOylation-deficient Coronin1C mutant forms cytoplasmic aggregates colocalizing with stress granules, a hallmark of neurodegenerative diseases, under neuronal differentiation conditions. We also demonstrate that Coronin1C SUMOylation works in synergy with Cdc42 GTPase towards actin-polymerization and promotes filopodia formation. In conclusion, we report that Coronin1C SUMOylation by SUMO1 in its carboxy-terminus is critical for cellular projections formation and neuronal differentiation.

cell biology↗

bioRxiv2025

Essential role for Nup43 in Drosophila fertility and spermiogenesis through Myosin VI-dependent actin cone assembly dynamics

Nuclear pore complexes, composed of nucleoporins (Nups), are critical for bidirectional nucleocytoplasmic transport. Studies on Nups have linked them with various cellular processes, including cell division, contributing significantly to organismal development. Intriguingly, Nup43, an integral member of Nup107 complex, is linked with premature ovarian insufficiency in humans. Here, we report that Nup43 is integral to the maintenance of Drosophila fertility. Both the females and males of Nup43 null mutant are sterile. In Nup43 mutants, embryonic development is halted at the first division stage, and males are sterile due to an arrest at the canoe stage of spermiogenesis. The nuclear elongation, shaping, and actin cone assembly steps of individualization complex formation are adversely affected, suspending sperm maturation. Expression through Nup43 transgene in Nup43 null mutants completely rescues spermiogenesis defects. Actin-based motor, Myosin VI (jar), interacts with Nup43 and rescues the actin cone assembly but not the sterility defects. We have uncovered a novel non-canonical function for Nup43 in Drosophila spermiogenesis, and propose that Nup43, along with jar, facilitates sperm individualization by promoting actin cone assembly.

developmental biology↗

bioRxiv2025

Heavy Metal Exposure Disrupts Electrotactic Behavior in Caenorhabditis elegans

Environmental toxicants such as heavy metals can profoundly impact organismal physiology, yet sensitive and rapid behavioral assays to quantify such effects are limited. Here, we employed a microfluidic-based electrotaxis assay to systematically evaluate the impact of chronic exposure to metal salts on the electrotactic swimming behavior of the nematode Caenorhabditis elegans. We report that exposure to Ag, Hg, MeHg, Cu, Mn, and Pb significantly alters electrotaxis speed. Notably, Cu required higher concentrations to induce phenotypes, whereas Ag and Hg disrupted behavior at lower doses. Three other metals (Ni, Fe, and Cd) did not elicit marked electrotaxis defects. Bioaccumulation analysis of Cu, Ag, Hg, and MeHg via ICP-MS revealed that MeHg was present in highest amount in worms. Consistent with this, MeHg showed the highest toxicity. We also found dopaminergic neurodegeneration in most metal-exposed animals, correlating with behavioral impairments. Although heat shock factor-1 (HSF-1) and its downstream target hsp-16.2 were induced by some metals, their expression did not consistently correlate with electrotaxis defects. Moreover, metal-induced impairments persisted even after recovery on toxin-free media, indicating possible irreversible damage. Our findings establish electrotaxis in C. elegans as a robust, non-invasive assay to assess neurobehavioral toxicity and demonstrate its utility for detecting sub-lethal impacts of environmental metals.

animal behavior and cognition↗

bioRxiv2024

D1 is a novel enhancer blocker protein that functions via multiple AT-rich simple sequence repeats

Simple sequence repeats (SSRs) are tandem repetitions of 1-6 bp DNA motifs at least 12 bp long. Certain length-enriched SSRs function as enhancers/silencers, enhancer blockers and barriers in a mammalian cell line. However, whether SSRs have similar cis-regulatory functions in vivo and their underlying mechanisms are unknown. To address this, we looked for SSR-binding proteins and investigated if SSRs function as enhancer blockers in vivo. We developed a novel Drosophila assay to assess enhancer blocker activity in vivo and to circumvent the low throughput and position effects seen in traditional enhancer blocker assays. Our assay uses endogenous vestigial gene enhancers, which, when blocked, result in easily scorable wing phenotypes increasing throughput. The attP-attB-based recombination system to integrate test fragments at specific site avoids positional effects. Furthermore, using EMSAs and DNA pull-downs followed by LC-MS/MS, we show that SSRs bind to proteins in a sequence-specific manner and identify 33 unique SSR-binding proteins. One of these proteins, D1, was enriched in several SSRs, viz., AAT14, AAAT10, AAAAT8, AATAT9, AAAG13 and AAAAG11. Using our novel vestigial in vivo enhancer blocker assay, we show for the first time that AT-rich SSRs function as enhancer blockers in a D1-dependent manner in Drosophila.

molecular biology↗

bioRxiv2024

Nup107 is a crucial regulator of Torso-mediated metamorphic transition in Drosophila melanogaster

Nuclear pore complexes (NPCs), composed of nucleoporins (Nups), affect nucleocytoplasmic transport, thus influencing cell division and gene regulation. Nup107 subcomplex members have been studied in housekeeping functions, diseases, and developmental disorders. We report a unique regulatory function for Nup107 in metamorphic transition during Drosophila development. RNAi-mediated Nup107-depleted larvae were arrested in the third-instar larval stage with no signs of pupariation. This lack of pupariation is primarily due to inhibited nuclear translocation and transcriptional activation by EcR. We demonstrate the involvement of Nup107 in the transcription of the Halloween genes, modulating ecdysone biosynthesis and the EcR pathway activation. The regulation of EcR-mediated metamorphosis by the receptor tyrosine kinase, torso, is well documented. Accordingly, overexpression of the torso and MAP-kinase pathway activator, rasV12, in the Nup107 depletion background rescues the phenotypes, implying that Nup107 is an epistatic regulator of Torso-mediated activation of EcR signaling during metamorphosis.

developmental biology↗

bioRxiv2023

Rho and a riboswitch regulate mntP expression evading manganese stress and membrane toxicity

The trace metal ion manganese in excess is toxic. Therefore, a small subset of factors tightly maintains its cellular level, among which an efflux protein MntP is the champion. Multiple transcriptional regulators and a manganese-dependent translational riboswitch regulate the MntP expression. As riboswitches are untranslated RNAs, they are often associated with the Rho-dependent transcription termination in bacteria. Here we demonstrate that Rho efficiently terminates transcription at the mntP riboswitch region. The addition of manganese activates the riboswitch, thereby restoring the coupling between transcription and translation to evade Rho-dependent transcription termination partially. Deletion of the riboswitch abolishes Rho-dependent termination and renders bacteria sensitive to manganese due to overexpression of mntP. The high mntP expression is associated with reactive oxygen species (ROS) production, slow growth, and cell filamentation phenotypes. We posit that manganese-dependent transcriptional activation in the absence of Rho-dependent termination leads to the observed toxicity arising from excessive MntP expression, a membrane protein. Thus, we identified a novel regulatory role of Rho in preventing membrane protein toxicity by terminating at the riboswitch element.

biochemistry↗

bioRxiv2023

Multigenerational inheritance of parasitic stress memory in Drosophila melanogaster

Organisms sense harmful environmental conditions and employ strategies to safeguard themselves. Moreover, they can communicate this experience to the next generation or beyond via non-DNA sequence-based mechanisms, referred to as intergenerational or transgenerational epigenetic inheritance, respectively. Using a specialist larval parasitoid, Leptopilina boulardi, and its host, Drosophila melanogaster, we show that the parental experience of parasitic stress results in an increased survivability of the immediate offspring of the host. Furthermore, we observe that the increased survivability in response to the parasitic stress is transmitted transgenerationally where the grandparents have been exposed to the parasitoid but not the parents. The increased survivability is primarily inherited through male parents, and at least one of the forms of the memory is better immune priming at larval stage. Our study suggests that the stress exposure during the pre-adult stage of the host has lifetime benefits for its progeny to deal with the future parasitic attack.

genetics↗

bioRxiv2023

DNMT3L interacts with Piwi and modulates the expression of piRNAs

The epigenetic modulator, DNMT3L, has been shown to be involved in nuclear reprogramming. Previous work from our laboratory had shown the accumulation and inheritance of epimutations across multiple generations when DNMT3L was ectopically expressed in transgenic DNMT3L Drosophila. Here, we show interaction of DNMT3L with Piwi, a member of a family of proteins known to perform their function through their association with piRNAs. Importantly, DNMT3L expression caused a significant alteration in the piRNA profile across multiple generations in transgenic Drosophila. As piRNAs are known to be passed on from one generation to another, we believe that the DNMT3L through its interaction with Piwi act to modify the inherited pool of piRNAs, which in turn allows accumulation of aberrant epigenetic modifications in subsequent generation. Furthermore, we show the interaction of DNMT3L with Histone H1, a non-core histone involved in higher order chromatin organisation. In light of these observations, it is proposed that in addition to its role in modulating core histone modifications, DNMT3L allows for inheritance of non-genetic information through its collaboration with Piwi, piRNAs and histone H1.

molecular biology↗

bioRxiv2023

Modeling of the Network Mediated by IL-36 Involved inPsoriasis

Pathogenesis of inflammatory, chronic and common skin disease Psoriasis involves immune cells, skin cells (keratinocytes) and cytokines secreted by them. Hyperproliferation and abnormal differentiation of keratinocytes are believed to be a hallmark of it. Roles of several cytokines such as TNF, IL-15, IL-17 and IL-23 in Psoriasis have been explored through mathematical/computational models as well as experimentally. However, the role of pro-inflammatory cytokine IL-36 is still elusive, especially in the case of General Pustular Psoriasis, a prevalent type of Psoriasis. To explore the role of that here, we construct a network embodying indirect cell-cell interactions of a few immune and skin cells mediated by IL-36 based on the existing knowledge. Further, we develop a mathematical model for the network and study the steady-state behaviour of that. Our results demonstrate that an increase in the level of IL-36 could lead to the hyper-proliferation of keratinocytes and, thus, Psoriasis. In addition, the analysis suggests that the plaque formation and progression of Psoriasis could occur via a gradual or switch-like increase in the population of keratinocytes. The switch-like increase would be due to the bistable behaviour of the network and could be used as a novel treatment strategy, as proposed and demonstrated earlier.

systems biology↗

bioRxiv2023

Atomic structure of wheat ribosome reveals unique features of the plant ribosomes

Ribosomes from plants have unique plant-specific features that may aid in rapid gene expression and regulation in response to changing environmental conditions due to their sessile nature. Here, we present high-resolution cryo-electron microscopy structures of the 60S and 80S ribosomes from wheat, a monocot staple crop plant (Triticum aestivum). We compare wheat ribosome with closely related ribosomes from a dicot plant and other eukaryotes from yeast to humans. While plant ribosomes have unique plant-specific rRNA modification (Cm1847) in peptide exit tunnel, Zinc-finger motif in eL34 is absent and uL4 is extended making an exclusive interaction network. We note striking differences in eL15-Helix 11 (25S) interaction network, eL6-Expansion segment 7 assembly and certain rRNA chemical modifications between monocot and dicot ribosomes. Among eukaryotic ribosomes, we observe that rRNA modification (Gm75) in 5.8S rRNA is highly conserved and a base flipping (G1506) in peptide exit tunnel, and these features are likely involved in sensing nascent peptide. Finally, we discuss importance of universal conservation of three consecutive rRNA modifications in all ribosomes for their interaction with A-site aminoacyl-tRNA.

plant biology↗

bioRxiv2023

Expansion of triplet nucleotide repeats in primates and other vertebrates: an evolutionary perspective

Triplet nucleotide repeat (TNR) expansion has been linked to more than 40 inheritable neurological, neuromuscular and neurodegenerative disorders. Increase in copy number beyond a threshold causes further rapid expansion of the repeats, leading to instability and disease via gain/loss of function, toxic RNA products or chromosome instability. An analysis of these repeat regions across vertebrates shows that these repeats have consistently either arisen late or have increased in copy number in vertebrates, most significantly in primates and particularly in humans. Many of the known diseases have neurological basis, suggests positive selection of these repeats for neuronal function. Late occurrence of the diseases implicates a lack of negative selection. This evolutionary trade-off, a higher neuronal capability at the cost of disease susceptibility, is further supported by the observation that most of the genes associated with TNR expansion diseases have neuronal function.

genomics↗

bioRxiv2023

Biochemical deconstruction and reconstruction of the Nuclear Matrix reveal the layers of nuclear organization

Nuclear Matrix (NuMat) is the fraction of the eukaryotic nucleus insoluble to detergents and high-salt extractions that manifests as a pan-nuclear fiber-granule network. NuMat consists of ribonucleoprotein complexes, members of crucial nuclear functional modules, and DNA fragments. Although NuMat captures the organization of non-chromatin nuclear space, very little is known about component organization within NuMat. To understand the organization of NuMat components, we subfractionated it with increasing concentrations of the chaotrope Guanidinium Hydrochloride (GdnHCl) and analyzed the proteomic makeup of the fractions. We observe that the solubilization of proteins at different concentrations of GdnHCl is finite and independent of the broad biophysical properties of the protein sequences. Looking at the extraction pattern of the Nuclear Envelope and Nuclear Pore Complex, we surmise that this fractionation represents easily-solubilized/loosely-bound and difficultly-solubilized/tightly-bound components of NuMat. Microscopic analyses of the localization of key NuMat proteins across sequential GdnHCl extractions of in situ NuMat further elaborate on the divergent extraction patterns. Furthermore, we solubilized NuMat in 8M GdnHCl and upon removal of GdnHCl through dialysis, en masse renaturation leads to RNA-dependent self-assembly of fibrous structures. The major proteome component of the self-assembled fibers comes from the difficultly-solubilized, tightly-bound component. This fractionation of the NuMat reveals different organizational levels within it which may reflect the structural and functional organization of nuclear architecture.

biochemistry↗

bioRxiv2022

Multigenerational effect of heat stress on the Drosophila melanogaster sperm proteome

The notion that genes are the sole units of heredity and that a barrier exists between soma and germline has been a major hurdle in elucidating the heritability of traits that were observed to follow a non-Mendelian inheritance pattern. It was only after the conception of epigenetics by Conrad Waddington that the effect of parental environment on subsequent generations via non-DNA sequence-based mechanisms, such as DNA methylation, chromatin modifications, non-coding RNAs and proteins, could be established, now referred to as multigenerational epigenetic inheritance. Despite growing evidence, the male gamete-derived epigenetic factors that mediate the transmission of such phenotypes are seldom explored, particularly in the model organism Drosophila melanogaster. Using the heat stress-induced multigenerational epigenetic inheritance paradigm in a widely used position-effect variegation line of Drosophila, named white-mottled, we have dissected the effect of heat stress on the sperm proteome in the current study. We demonstrate that multiple successive generations of heat stress at the early embryonic stage results in a significant downregulation of proteins associated with a diverse set of functions, such as translation, chromatin organization, microtubule-based processes, and generation of metabolites and energy, in the sperms. Based on our findings, we propose chromatin-based epigenetic mechanisms, a well-established mechanism for multigenerational effects, as a plausible way of transmitting heat stress memory via the male germline in this case. Moreover, we show that despite these heat stress-induced changes, the life-history traits, such as reproductive fitness and stress tolerance of the subsequent generations, are unaffected, probing the evolutionary relevance of multigenerational epigenetic effects.

developmental biology↗

bioRxiv2022

An intrinsic alkalization circuit turns on mntP-riboswitch under manganese stress in Escherichia coli

The trace metal manganese in excess affects iron-sulfur cluster and heme-protein biogenesis eliciting cellular toxicity. The manganese efflux protein MntP is crucial to evading manganese toxicity in bacteria. Recently, two Mn-sensing riboswitches upstream of mntP and alx in Escherichia coli have been reported to mediate the upregulation of their expression under manganese shock. As the alx-riboswitch is also responsive to alkaline shock administered externally, it is intriguing whether mntP-riboswitch is also responsive to alkaline stress. Furthermore, how both manganese and alkaline pH simultaneously regulate these two riboswitches under physiological conditions is a puzzle. Using multiple approaches, we show that manganese shock activated glutamine synthetase (GlnA) and glutaminases (GlsA and GlsB) to spike ammonia production in E. coli. The elevated ammonia intrinsically alkalizes the cytoplasm. We establish that this alkalization under manganese stress is crucial for attaining the highest degree of riboswitch activation. Additional studies showed that alkaline pH promotes a 17 to 22-fold tighter interaction between manganese and the mntP-riboswitch element. Our study uncovers a physiological linkage between manganese efflux and pH homeostasis that mediates enhanced manganese tolerance. Significance statementRiboswitch RNAs are cis-acting elements that can adopt alternative conformations in the presence or absence of a specific ligand(s) to modulate transcription termination or translation initiation processes. In the present work, we show that how manganese and alkaline pH both are necessary for maximal mntP-riboswitch activation to mitigate the manganese toxicity. This study bridges the gap between earlier studies that separately emphasize the importance of alkaline pH and manganese in activating the riboswitches belonging to the yybP-ykoY-family. This study also ascribes a physiological relevance as to how manganese can rewire cellular physiology to render cytoplasmic pH alkaline for its homeostasis.

biochemistry↗

bioRxiv2022

Bird assemblages in a peri-urban landscape in eastern India

Across the tropics, human settlements and agricultural lands have rapidly replaced the natural habitat; however, our understanding of the biodiversity value in such human-dominated landscapes is limited. To study the effects of heterogeneous habitats on bird diversity in the peri-urban landscape, we surveyed four different habitat types (residential areas, cropland and wasteland, water bodies, and sal forest) from February 2018 to January 2019. During the survey, 117 bird species were recorded belonging to 48 families and 98 genera in the study area. We observed that cropland and wasteland harboured rich avian diversity, among other sites. Both the Non-parametric Multidimensional Scale (NMDS) and one-way ANOVA Significant Test showed significant variations in bird species composition among habitats. Our study could be helpful in future conservation and management of the birds diversity to make peri-urban habitats more favorable.

ecology↗

bioRxiv2022

Sofosbuvir inhibits the RNA dependent RNA polymerase activity of non-Structural protein 5 from Kyasanur forest disease virus

Kyasanur forest disease is a neglected zoonotic disease caused by a single-stranded RNA-based flavivirus, the incidence of which was first recorded in 1957 in the Southern part of India. Kyasanur forest disease virus is transmitted to monkeys and humans through the infected tick bite of Haemophysalis spinigera. Kyasanur forest disease is a febrile illness, which in severe cases, results in neurological complications leading to mortality. The current treatment regimens are symptomatic and supportive, and no targeted therapies are available for this disease. In this study, we evaluated the ability of FDA-approved drugs sofosbuvir (and its active metabolite) and Dasabuvir to inhibit the RNA-dependent RNA polymerase activity of NS5 protein from the Kyasanur forest disease virus. NS5 protein containing the N-terminal methyl transferase domain and C-terminal RNA-dependent RNA polymerase domain was expressed in Escherichia coli, and RNA-dependent RNA polymerase activity was demonstrated with the purified protein. The RNA-dependent RNA polymerase assay conditions were optimized, followed by the determination of apparent Km,ATP to validate the enzyme preparation. Half maximal-inhibitory concentrations against RNA-dependent RNA polymerase activity were determined for Sofosbuvir and its active metabolite. Dasabuvir did not show detectable inhibition with the tested conditions. This is the first demonstration of the inhibition of RNA-dependent RNA polymerase activity of NS5 protein from the Kyasanur forest disease virus with small molecule inhibitors. These initial findings can potentially facilitate the discovery and development of targeted therapies for treating Kyasanur forest disease.

biochemistry↗

bioRxiv2021

DiviSSR: Simple arithmetic for efficient identification of tandem repeats

Numerical or vector representations of DNA sequences have been applied for identification of specific sequence characteristics and patterns which are not evident in their character (A, C, G, T) representations. These transformations often reveal a mathematical structure to the sequences which can be captured efficiently using established mathematical methods. One such transformation, the 2-bit format, represents each nucleotide using only two bits instead of eight for efficient storage of genomic data. Here we describe a mathematical property that exists in the 2-bit representation of tandemly repeated DNA sequences. Our tool, DiviSSR (pronounced divisor), leverages this property and subsequent arithmetic to achieve ultrafast and accurate identification of tandem repeats. DiviSSR can process the entire human genome in [~]30s, and short sequence reads at a rate of >1 million reads/s on a single CPU thread. Our work also highlights the implications of using simple mathematical properties of DNA sequences for faster algorithms in genomics. AvailabilityDiviSSR can be installed directly using python pip. The source code and documentation of DiviSSR are available at https://github.com/avvaruakshay/divissr.git.

bioinformatics↗