Role of Cellular Membranes in Controlling Micro RNA Mediated Co-ordinated Gene Expression

MicroRNAs are ~22 nt. regulatory RNAs that bind to 3’ UTR of target mRNA and regulate the expression by translational repression or transcriptional inactivation. Previously it has been reported there exists a target mRNA dependent miRNA biogenesis where target mRNA could regulate its cognate miRNA biogenesis by modulating Dicer processivity. It is well known that multiple miRNAs share the common 3’ UTR of the same target mRNA. So I was curious to check the additive level of regulation in “target mRNA dependent miRNA biogenesis” on other miRNAs that share binding sites on the same 3`UTR of target mRNA. My aim was to dissect the molecular manifestation of this “cooperative regulation” and its relevance in normal cellular physiology. Earlier It was observed there exists a target mRNA dependent miRNA biogenesis where one target mRNA could regulate its cognate miRNA biogenesis by modulating Dicer processivity. I was curious how this phenomenon affects biogenesis of other miRNAs on the same 3`UTR. I could observe miRNA family with a higher number of binding sites (miRNA 1) influences the biogenesis of adjacent groups of miRNAs (miRNA 2) that shares the common 3’ UTR. Along with, I could also observe repression of secondary mRNAs (mRNA B) that bear sites for miRNA2 (but not for miRNA1) is co-ordinately regulated with miRNA1 abundance inside the cells. Using a reporter based model in endotoxin-stress induced TLR4-activation module, corroborate my findings to propose “target dependent cooperative biogenesis of miRNAs' ' phenomenon inside the mammalian cells. A computational module was developed to predict regulatory miRNA and their network under different physiological contexts. Interestingly again, many of those predicted cooperative miRNAs and its’ secondary target has been revalidated biochemically that exhibits the same trend, strengthening the proposed hypothesis. I could observe coordinated regulation of miRNAs resulting in alleviating 5 bacterial endotoxin-induced pro-inflammatory response. My observations suggest "target dependent cooperative biogenesis of miRNAs (TDCB)” added an additional layer of finetuning of signalling molecules on endotoxin-responsive murine macrophages to re-establish cellular homeostasis. Additionally I have also shown mechanistically co-operative biogenesis of miRNAs on macrophages play an important role to combat pro-inflammatory response. Along with I have also explored the cellular compartmentalization of this phenomenon. Previous reports suggest polysome attached with rER serves as the nucleation site for miRNP assembly and miRNA mediated target mRNA repression. Here, I have observed the differential compartmentalization of miRNAs and target mRNAs regulated by target dependent cooperative biogenesis on those cellular compartments. The other part of work has been concentrated on the study on importance of cellular membranes on miRNA biogenesis and rER targeting of target mRNA and how this influences the fate of miRNA. There I have observed reduced compartmentalization of de novo formed miRNAs on rER or polysomes in defective mitochondria containing cells along with increased retention of its target mRNAs. I have also observed defective target mRNA dependent cognate miRNA biogenesis in an amino acid starved and refed hepatic cells. Increased retention of microsomal target mRNAs in mitochondria-ER detethered cells due to impaired recycling of miRNP components has been also observed in Mfn2 negative cells. I have also found a defective intracellular trafficking in growth retarded senescent mammalian cells having impaired mitochondrial potential and dynamics. Similar to what happens in senescent cells, Uncoupling Protein 2 mediated depolarization of mitochondrial membrane potential results in progressive sequestration of miRNAs with polysomes. Mitochondrial detethering of endoplasmic reticulum, a phenomenon also evident in mitochondria depolarized cells, found to be responsible for defective compartmentalization of 6 translation initiation factor eIF4E to ER attached polysomes. It causes retarded translation process accompanied by enhanced retention of miRNAs and target mRNAs with rER attached polysomes that resulted in reduced intracellular trafficking. In subsequent experiments I have identified a reduced activity of mTORC1 in mitochondria defective cells to cause reduced phosphorylation of eIF4E-BP1 on microsomes to cause retarded eIF-4E targeting to ER attached polysome. Cumulatively, these data suggest intricate involvement of mitochondrial membrane potential and dynamics to determine stability of miRNAs in mammalian cells by affecting subcellular locations and export of miRNPs. These data suggest how mitochondrial membrane potential and dynamics, by targeting mTORC1 activity and compartmentalization, determine subcellular localization of miRNPs. This infers mitochondrial detethering of rER may directly cause lowering of miRNA turnover by targeting the initiation phase of translation caused by poor shuttling of cap binding protein eIF4E from cytoplasmic pool to the rER associated domain

Regulation Of Cell Signaling By Inter-Organeller Interaction In Neuronal And Non-Neuronal Cells: Effect On MicroRNA Mediated Gene Regulation

Uponexposuretoamyloid-β oligomers(Aβ1–42), glialcellsstart expressingproinflammatorycytokines,despiteanincreaseinlevels of repressivemicroRNAs(miRNAs).Exploringthemechanismof thispotentialimmunityoftargetcytokinemRNAsagainstrepressive miRNAsinamyloid-β-exposedglialcells,wehaveidentifieddifferential compartmentalizationofrepressivemiRNAsinglialcellsthatexplains thisaberrantmiRNAfunction.InAβ1–42-treatedcells,whereastarget mRNAswerefoundtobeassociatedwithpolysomesattachedto endoplasmicreticulum(ER),themiRNAribonucleoproteincomplexes (miRNPs)werefoundtobepresentpredominantlywithendosomes thatfailedtorecycletoER-attachedpolysomes,preventingrepression of mRNAtargets.Aβ1–42 oligomers,bymaskingRab7aproteinson endosomalsurfaces,affectedRab7ainteractionwithRab-interacting lysosomalprotein(RILP),restrictingthelysosomaltargetingand recyclingofmiRNPs.RNA-processingbody(P-body)localizationof themiRNPswasfoundtobeenhancedinamyloid-β-treatedcellsas a consequenceofenhancedendosomalretentionofmiRNPs. Interestingly,depletionofP-bodycomponentspartlyrescuedthe miRNAfunctioninglialcellsexposedtoamyloid-β andrestrictedthe excesscytokineexpression

Dynamics and electrostatics define an allosteric druggable site within the receptor-binding domain of SARS-CoV-2 spike protein

The pathogenesis of the SARS-CoV-2 virus initiates through recognition of the angiotensin-converting enzyme 2 (ACE2) receptor of the host cells by the receptor-binding domain (RBD) located at the spikes of the virus. Here, using molecular dynamics simulations, we have demonstrated the allosteric crosstalk within the RBD in the apo- and the ACE2 receptor-bound states, revealing the contribution of the dynamics-based correlated motions and the electrostatic energy perturbations to this crosstalk. While allostery, based on correlated motions, dominates inherent distal communication in the apoRBD, the electrostatic energy perturbations determine favorable pairwise crosstalk within the RBD residues upon binding to ACE2. Interestingly, the allosteric path is composed of residues which are evolutionarily conserved within closely related coronaviruses, pointing toward the biological relevance of the communication and its potential as a target for drug development