Biology subjects

Srivastava, J.

Publications and source records attributed to Srivastava, J..

9 recordsLinked to original sources

bioRxiv2026

FOXO3 regulated MIR503HG safeguards cellular quiescence by modulating PI3K/Akt pathway via miR-508/PTEN axis

Long noncoding RNAs (LncRNAs) have emerged as a class of important regulatory ncRNAs and are known to fine-tune numerous cellular processes including proliferation, differentiation and development; however, their role in quiescence still remains largely unexplored. A miRNA host gene lncRNA, MIR503HG, has been reported to play important role in cancer development. Here, we demonstrate the role of MIR503HG lncRNA in regulating cellular quiescence. MIR503HG displays elevated levels in human diploid fibroblasts induced to undergo quiescence. Depletion of MIR503HG in HDFs affects the entry of cells into quiescence but has no effect on cell cycle progression, suggesting its role in quiescence attainment and/or maintenance. Additionally, MIR503HG depletion led to a drastic decrease in the levels of miR508 target, PTEN with a concomitant increase in pAkt levels, indicating its role in negative regulation of miR508. Further, we demonstrate that the lncRNA MIR503HG regulates PTEN levels by acting as a ceRNA for miR508 to maintain cellular quiescence. Our studies illustrate that MIR503HG can function synergistically with miR503 to maintain cells under quiescence and both the miRNA-HG and the miRNA encoded by its gene locus synergistically control the same biological process in different ways by regulating different downstream genes.

cell biology↗

bioRxiv2026

lncRNA NORM is essential for proper chromosome segregation through the Plk1-Bub1 and Nsun2 axis.

The cell cycle comprises different phases and is a tightly regulated process at the molecular level. During the cell cycle, two key events occurred: DNA duplication during the S phase and chromosome segregation during mitosis. Accurate cell cycle progression, achieved through faithful chromosome segregation, is essential for maintaining cell fidelity. Long noncoding RNAs are a subclass of noncoding RNA that are longer than 200 bp and form RNA protein complexes (RNPs) to regulate various biological processes. Herein, we demonstrate that lncRNA NORM is involved in regulating the cell cycle by maintaining proper chromosome segregation. NORM exhibited G2 phase-specific expression, and the depletion of NORM resulted in a significant G2/M arrest. NORM-depleted cells failed to progress in mitosis and showed defects in chromosome segregation. We further demonstrated that NORM binds to proteins such as Plk1 and Nsun2. Depletion of NORM hindered the interaction between Plk1 and Bub1, resulting in reduced kinetochore localization of Plk1 during prometaphase. Our results also show that the depletion of NORM affects the binding of Nsun2 protein to CDK1 mRNA and, consequently, the stabilization of CDK1 at the protein level. Altogether, our results demonstrate that NORM regulates chromosome segregation by mediating the interaction between Plk1 and Bub1.

cell biology↗

bioRxiv2024

Regulatory plasticity of the human genome

Evolutionary turnover in non-coding regions has driven phenotypic divergence during past speciation events and continues to facilitate environmental adaptation through variants. We used a deep learning model to identify the substrates of regulatory turnover using genome wide mutations mimicking three evolutionary pathways: recent history (human-chimp substitutions), modern population (human population variation), and mutational susceptibility (random mutations). We observed enhancer turnover in approximately 6% of the whole genome, with more than 80% of the novel activity arising from repurposing of enhancers between cell-types. Frequency of turnover in a cell-type is remarkably similar across the three pathways, despite only [~]19% overlap in the source regions. The enhancers predisposed to turnover display reduced evolutionary constraints and are depleted in GWAS variants. Most of these occur within 100kb of a gene, with the highest turnover occurring near neurodevelopmental genes including CNTNAP2, NPAS3, and AUTS2. Flanking enhancers of these genes undergo high turnover irrespective of the mutational model pathway, suggesting a high plasticity in neurocognitive evolution. Based on susceptibility to random mutations, these enhancers were identified as vulnerable by nature and feature a higher abundance of cell type-specific transcription factor binding sites (TFBSs). Our findings suggest that enhancer repurposing within vulnerable loci drives regulatory innovation while keeping the core regulatory networks intact.

genomics↗

bioRxiv2024

Altered Expression of microRNAs Implicated in Hematopoietic Dysfunction in the Extracellular Vesicles of Bone Marrow-Mesenchymal Stromal Cells in Aplastic Anemia

Recently, we have reported that extracellular vesicles (EVs) from the bone marrow mesenchymal stromal cells (BM-MSC) of aplastic anemia (AA) patients inhibit hematopoietic stem and progenitor cell (HSPC) proliferative and colony-forming ability and promote apoptosis. One mechanism by which AA BM-MSC EVs might contribute to these altered HSPC functions is through microRNAs (miRNAs) encapsulated in EVs. However, little is known about the role of BM-MSC EVs derived miRNAs in regulating HSPC functions in AA. Therefore, we performed miRNA profiling of EVs from BM-MSC of AA (n=6) and normal controls (NC) (n=6), to identify differentially expressed miRNAs carried in AA BM-MSC EVs. DEseq2 analysis identified 34 significantly altered mature miRNAs in AA BM-MSC EVs. Analysis of transcriptome dataset of AA HSPC genes identified that 235 differentially expressed HSPC genes were targeted by these 34 EV miRNAs. The pathway enrichment analysis of 235 HSPC genes revealed their involvement in pathways associated with cell cycle, proliferation, apoptosis, and hematopoiesis regulation, thus highlighting that AA BM-MSC EV miRNAs could potentially contribute to impaired HSPC functions in AA.

molecular biology↗

bioRxiv2021

Clues to reaction specificity in PLP-dependent fold type I enzymes of monosaccharide biosynthesis

Novel functions can emerge in an enzyme family while conserving catalytic mechanism, motif or fold. PLP-dependent enzymes have evolved into seven fold-types and catalyse diverse reactions using the same mechanism for the formation of external aldimine. Nucleotide sugar aminotransferases (which will be henceforth referred to as aminotransferases) belong to fold type I and mediate the biosynthesis of several monosaccharides. They use diverse substrates but are highly selective to the C3 or C4 carbon to which amine group is transferred. Profile HMMs were able to identify aminotransferases but could not capture reaction specificity. A search for discriminating features led to the discovery of sequence motifs that are located near the pyranose binding site suggesting their role in imparting reaction specificity. Using a position weight matrix for this motif, we were able to assign reaction specificity to a large number of aminotransferases. Inferences from this analysis set way for future experiments that can shed light on mechanisms of functional diversification in nucleotide sugar aminotransferases of fold type I.

bioinformatics↗

bioRxiv2021

A cost-effective and efficient approach for generating and assembling reagents for conducting real-time PCR

Real-time PCR is a widely used technique for quantification of gene expression. However, commercially available kits for real-time PCR are very expensive. The ongoing coronavirus pandemic has severely hampered the economy in a number of developing countries, resulting in a reduction in available research funding. The fallout of this will result in limiting educational institutes and small enterprises from using cutting edge biological techniques such as real-time PCR. Here, we report a cost-effective approach for preparing and assembling cDNA synthesis and real-time PCR mastermixes with similar efficiencies as commercially available kits. Our results thus demonstrate an alternative to commercially available kits.

molecular biology↗

bioRxiv2021

Searching sequence databases for functional homologs using profile HMMs: how to set bit score thresholds?

MotivationUniProt and BFD databases together have 2.5 billion protein sequences. A large majority of these proteins have been electronically annotated. Automated annotation pipelines, vis-a-vis manual curation, have the advantage of scale and speed but are fraught with relatively higher error rates. This is because sequence homology does not necessarily translate to functional homology, molecular function specification is hierarchic and not all functional families have the same amount of experimental data that one can exploit for annotation. Consequently, customization of annotation workflow is inevitable to minimize annotation errors. ResultsWe discuss possible ways of customizing the search of sequence databases for functional homologs using profile HMMs. Choosing an optimal bit score threshold is a critical step in the application of HMMs, which is illustrated using four Case Studies; the single domain nucleotide sugar 6-dehydrogenase and lysozyme-C families, and SH3 and GT-A domains which are typically found as a part of multi-domain proteins. We also discuss the limitations of using profile HMMs for functional annotation and suggests some possible ways to partially overcome such limitations. Supplementary informationSupplementary_material containing Figures S1-S7 and Tables S1 and S2 Supplementary_dataset.xlsx

bioinformatics↗

bioRxiv2020

GlycoPathDB: A database of monosaccharide biosynthesis pathways

A distinctive feature of glycans vis-a-vis proteins and nucleic acids is its structural complexity which arises from the huge repertoire of monosaccharides, isomeric linkages and branching. Around 70 monosaccharides have so far been discovered in natural glycans and the pathway for the biosynthesis of 57 of these have been experimentally characterized. However, there is no common platform with a comprehensive information of monosaccharide pathways and enzymes. We have gathered 542 experimentally characterized enzymes of these pathways from literature and set up a first of its kind database called GlycoPathDB (http://www.bio.iitb.ac.in/glycopathdb/). Annotations such as the reaction catalysed, substrate specificity, biosynthesis pathway and PubMed IDs are provided for all the enzymes in the database. Sequence homologs of the experimentally characterized enzymes found in nearly 13,000 completely sequenced genomes from Bacteria and Archaea have also been included in the database. This platform will help in deduction of evolutionary relationships among enzymes such as aminotransferases, nucleotidyltransferases, acetyltransferases and SDR family enzymes. It can also facilitate experimental studies such as direct enzyme assays to validate putative annotations, establish structure-function relationship, expression profiling to determine the function, determine the phenotypic consequences of gene knock-out/knock-in and complementation studies.

bioinformatics↗

bioRxiv2020

Is there a universal glycan alphabet?

Several monosaccharides constitute naturally occurring glycans but it is uncertain if they constitute a universal set like the alphabets of proteins and DNA. Based on the available experimental observations, it is hypothesized herein that the glycan alphabet is not universal. Data on the presence / absence of pathways for the biosynthesis of 55 monosaccharides in 12939 completely sequenced archaeal and bacterial genomes are presented in support of this hypothesis. Pathways were identified by searching for homologs of biosynthesis pathway enzymes. Substantial variations are observed in the set of monosaccharides used by organisms belonging to the same phylum, genera and even species. Monosaccharides are grouped as Common, Less Common and Rare based on their prevalence in Archaea and Bacteria. It is observed that fewer enzymes suffice to biosynthesize the Common group. It appears that the Common group originated before the formation of three domains of life. In contrast, the Rare group are confined to a few species in a few phyla, suggesting that they evolved much later. Fold conservation, as observed in aminotransferases and SDR superfamily members involved in monosaccharide biosynthesis, suggests neo- and sub-functionalization of genes leading to the formation of Rare group monosaccharides. Non-universality of the glycan alphabet begets questions about the role of different monosaccharides in determining an organisms fitness. Impact statementCarbohydrates, nucleic acids and proteins are important classes of biological macromolecules. The universality of DNA, RNA and protein alphabets has been established beyond doubt. However, the universality of glycan alphabet is unknown primarily because of the challenges associated with the elucidation of glycan structures. This has precluded a comprehensive investigation of glycan alphabet. To address this challenge, we have identified the prevalence of 55 monosaccharide biosynthesis pathways in 12939 completely sequenced archaeal and bacterial genomes by searching for homologs of biosynthesis pathway enzymes using HMM profiles, and in a few cases, BLASTp. This revealed that the glycan alphabet is highly variable; in fact, significant differences are found even among different strains of a species. Possible implications of this variability may be significant in understanding the evolution of Archaea and Bacteria in diverse and competitive environments. Factors that drive the choice of monosaccharides used by an organism need to be investigated, and will be of interest in understanding host-pathogen interactions. Additionally, the knowledge of glycan alphabet can be employed for structural characterization / validation of glycans inferred using mass spectrometry. Knowledge of unique monosaccharides and biosynthetic enzymes can also be used as novel drug targets against human pathogens. Data summaryThe curated set of proteins used in this study, with domain assignment, is listed in supplementary_data.xlsx. Corresponding 396 references with evidence of experimental characterization are included in supplementary material. Results of genome scan which include predictions of monosaccharides as well as the biosynthesis pathway enzymes is available at http://www.bio.iitb.ac.in/glycopathdb/ including the aforementioned information. Python script used to scan genomes to search for monosaccharide biosynthesis pathways are available on request.

genomics↗