Biology subjects

Parashar, V.

Publications and source records attributed to Parashar, V..

4 recordsLinked to original sources

bioRxiv2025

EVRCEPT: EV RNA Cargo Enrichment Prediction Tool to predict enrichment of RNA into Extracellular Vesicles

Extracellular vesicles (EVs) are small membrane-bound vesicles that are released by most cells. EVs have been shown to transport molecules including proteins and various types of RNAs between cells of even different types. Furthermore, EV RNAs are shown to modulate gene expression in physiological and pathological conditions in recipient cells which can be utilized in therapeutics by engineering cells to enrich RNA of interest in EVs. However, how specific RNA species are enriched in EVs is a long-standing question in the field. Here, we used sequence features of RNAs to predict its enrichment in EVs. These features include length, nucleotide and dinucleotide frequencies, secondary structure information, number of exons, coding probability for non-coding RNAs as well as RNA binding protein (RBP) motifs. The model achieved a performance (AU-ROC: 90%, 77%) for circRNAs and mRNAs, respectively. Here, we present a web tool called, EV RNA Cargo Enrichment Prediction Tool (EVRCEPT), that allows users to predict likelihood of input RNA to be enriched into EVs. This tool will also provide the list of RBPs that are likely to interact with the input RNA and works with both linear and circular RNAs. This webtool, which is freely accessible at https://euler.dbi.udel.edu/evrcept, will help understand extracellular RNA transport and guide the design of therapeutic RNAs to maximize their incorporation in EVs towards targeted personalized medicine.

bioinformatics↗

bioRxiv2025

Structural Insights into Allosteric Regulation of GdpP: A Conformationally Dynamic Phosphodiesterase

The phosphodiesterase GdpP is a central regulator of the bacterial second messenger c-di-AMP and a key driver of antibiotic resistance in pathogenic Firmicutes. GdpP integrates environmental signals through its sensory PAS domain to control its C-terminal catalytic domain activity, but the molecular basis for this allosteric communication has remained unknown due to the lack of structural data for the complete cytosolic region. Here, we present the first Cryo-EM structures of the cytosolic region of Streptococcus mutans GdpP (SmGdpP74) in multiple conformational states, revealing a sophisticated tetrameric architecture that enables asymmetric catalytic regulation. Our structural and functional analyses demonstrate that SmGdpP74 operates through substrate-induced conformational transition, with the DHHA1 domain interface serving as the primary determinant of asymmetric catalysis. The non-canonical GGDEF domain functions as a tetrameric scaffolding hub that positions the DHH-DHHA1 catalytic domains, representing the first detailed example of a GGDEF domain repurposed to stabilize heterologous catalytic domains. We identify a flexible GGDEF-DHH linker as a critical coupling element that transmits conformational signals between domains, with the conserved KRSR motif acting as a molecular switch for heme-mediated inhibition. Additionally, the flexibility of this linker is essential for the enzymes catalytic activity. Based on these findings, we propose a comprehensive mechanistic model where SmGdpP74 integrates substrate availability with conformational changes for efficient hydrolysis and allosteric control of its enzymatic activity through the PAS domain. These structural insights provide a foundation for rational drug design targeting allosteric regulatory mechanisms in GdpP, potentially offering new approaches to combat antibiotic resistance in pathogenic Firmicutes.

molecular biology↗

bioRxiv2024

Defining the parameters for sorting of different RNA cargo into Extracellular vesicles

Extracellular vesicles (EVs) are small particles that are released by cells and mediate cell-cell communication by transferring bioactive molecules. RNA cargo of EVs, including coding and non-coding RNAs, can change the behavior of recipient cells, affecting processes like gene expression, proliferation, and apoptosis. Circular RNAs (CircRNA) are a newly appreciated class of regulatory RNAs that are stable, resistant to degradation and have been shown to be enriched in EVs. They play key roles in gene regulation and are also emerging as promising biomarkers for disease diagnosis. While microRNAs (miRNAs) are the most well studied RNA cargo of EVs, very little is known about the mechanisms of enrichment of circRNAs as well as long linear RNAs. Here, we take a comprehensive genome-wide approach to investigate the role of GC%, size, exon count, structuredness and coding potential, in the sorting and enrichment of circular and long linear RNAs into EVs. We found that size and structuredness had a significant role in enriching RNAs into EVs which was consistent across all classes of RNAs. Furthermore, we found that structuredness could explain the relative enrichment of circRNAs over their linear counterparts. These results were validated on existing public databases of circular and linear RNAs in EVs. By identifying and analyzing these factors, we aim to better understand the complex mechanisms behind EV-mediated RNA transfer and its impact on cell communication in both health and disease. This mechanistic understanding of RNA enrichment in EVs is crucial for engineering EVs with selective RNA cargo.

cell biology↗

bioRxiv2020

Investigations of dimethylglycine (DMG), glycine betaine and ectoine uptake by a BCCT family transporter with broad substrate specificity in Vibrio species.

Fluctuations in osmolarity are one of the most prevalent stresses to which bacteria must adapt, both hypo- and hyper-osmotic conditions. Most bacteria cope with high osmolarity by accumulating compatible solutes (osmolytes) in the cytoplasm to maintain the turgor pressure of the cell. Vibrio parahaemolyticus, a halophile, utilizes at least six compatible solute transporters for the uptake of osmolytes: two ABC family ProU transporters and four betaine-carnitine-choline transporter (BCCT) family transporters. The full range of compatible solutes transported by this species has yet to be determined. Using an osmolyte phenotypic microarray plate for growth analyses, we expanded known osmolytes used by V. parahaemolyticus to include N-N dimethylglycine (DMG) amongst others. We showed that V. parahaemolyticus requires a BCCT transporter for DMG uptake, carriers that were not known to transport DMG. Growth pattern analysis of four triple-bccT mutants, possessing only one functional BCCT, indicated that BccT1 (VP1456), BccT2 (VP1723), and BccT3 (VP1905) transported DMG, which was confirmed by functional complementation in E. coli strain MKH13. BccT1 was unusual in that it could uptake both compounds with methylated head groups (glycine betaine (GB), choline and DMG) and cyclic compounds (ectoine and proline). Bioinformatics analysis identified the four coordinating residues for glycine betaine in BccT1. In silico modelling analysis demonstrated that glycine betaine, DMG, and ectoine docked in the same binding pocket in BccT1. Using site-directed mutagenesis, we showed that a strain with all four resides mutated resulted in loss of uptake of glycine betaine, DMG and ectoine. We showed three of the four residues were essential for ectoine uptake whereas only one of the residues was essential for glycine betaine uptake. Overall, we have demonstrated that DMG is a highly effective compatible solute for Vibrio species and have elucidated the amino acid residues in BccT1 that are important for coordination of glycine betaine, DMG and ectoine transport. ImportanceVibrio parahaemolyticus possesses at least six osmolyte transporters, which allow the bacterium to adapt to high salinity conditions. In this study, we identified several novel osmolytes that are utilized by V. parahaemolyticus. We demonstrated that the compound dimethylglycine (DMG), which is abundant in the marine environment, is a highly effective osmolyte for Vibrio species. We determined that DMG is transported via BCCT-family carriers, which have not been shown previously to uptake this compound. BccT1 was a carrier for glycine betaine, DMG and ectoine and we identified the amino acid residues essential for coordination of these compounds. The data suggest that for BccT1, glycine betaine is more easily accommodated than ectoine in the transporter binding pocket.

microbiology↗