Biology subjects

Bhattacharyya, S. N.

Publications and source records attributed to Bhattacharyya, S. N..

17 recordsLinked to original sources

bioRxiv2026

miRNA Sensor HuR Compartmentalizes Ago2-Uncoupled miRNAs to Lipid Droplets to Buffer miRNA Activity in Mammalian Cells

miRNA activity must be optimized to ensure target gene expression in mammalian cells in response to specific needs. How and when miRNA activity is regulated remains an open question in gene expression regulation, with limited information on how cellular machinery senses miRNA levels to regulate miRNA expression and activity. Because miRNAs are stable molecules that can be reversibly used, the existence of a miRNA-sensing mechanism in mammalian cells has been anticipated. With ectopic expression of miRNAs in mammalian cells, we found a dose-dependent miRNA buffering mechanism in which miRNA export and its storage on lipid droplets are essential for miRNA activity optimization in mammalian cells. We found that the miRNA-binding protein HuR, known for its role in miRNA export, has a dual function in mammalian cells. HuR uncouples miRNAs from Ago2 to facilitate association of lipid droplets with nonfunctional miRNAs, a process that gets augmented in cells with high lipid droplet content and restricted extracellular export. Thus, HuR acts as a miRNA sensor, and the optimal activity and abundance of HuR regulate miRNA storage on lipid droplets or its export to maintain cellular miRNA homeostasis, thereby preventing detrimental effects of an excess miRNA pool in mammalian cells. Thus, targeting miRNA export or lipid droplet association is an important strategy for buffering cellular miRNA levels. ImportanceTo ensure that target gene expression in mammalian cells is appropriately regulated, miRNA activity needs to be carefully fine-tuned. Although miRNAs are stable and can be reused, the precise mechanisms by which their levels are sensed and regulated remain unclear. Our research uncovered a fascinating dose-dependent miRNA buffering process that involves export and storage of miRNAs on lipid droplets--essential steps for precise regulation. We also found that HuR, a protein that facilitates miRNA export, plays a key role by uncoupling miRNAs from Ago2 and promoting their association with lipid droplets in cells with abundant lipid droplets and limited extracellular export capacity. Overall, HuR acts as a miRNA sensor, helping to balance miRNA storage and export to maintain cellular homeostasis and prevent the harmful effects of miRNA overactivity.

cell biology↗

bioRxiv2025

Isolation and Quantification of mRNAs from Subcellular Phase-Separated Structures from Detergent Permeabilized Brain Cells

Post-transcriptional regulation by RNA processing bodies, also known as P-bodies (PBs), is crucial for mRNA translation, localization, and stability in various animal cells, including neurons and glial cells. PBs facilitate spatial regulation of protein synthesis, influencing differentiation and synaptic function. Understanding mRNA dynamics in PBs is vital and is suspected to be impaired in neurodegenerative disorders. We developed a detergent-based method to isolate phase-separated P-bodies (PBs) free from cytosolic factors and RNAs, which allows us to examine their mRNA content under altered conditions. Using neuronal and glial cell models, we investigated the impact of A{beta}-oligomers on PB-associated mRNAs. In neurons, exposure to amyloid proteins disrupted the release of neuronal differentiation-related mRNAs, hindering their translation. Glial cells exhibited increased levels of cytoplasmic pro-inflammatory cytokine mRNAs after amyloid treatment, as they escaped from RNA processing bodies (PBs), ensuring enhanced translation. These findings underscore the dual role of PBs in controlling mRNA dynamics across cell types and illustrate how amyloid-induced stress may differently affect PB-mediated post-transcriptional control in neurons and astroglia. This method provides a platform for studying the mechanism and quantifying sub-organellar mRNA localization, as well as its effects on altered gene expression caused by amyloid proteins. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/669016v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@335e76org.highwire.dtl.DTLVardef@7ecf8dorg.highwire.dtl.DTLVardef@16c375dorg.highwire.dtl.DTLVardef@1a6c086_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA detergent-based differential solubilization of RNAs from PBs in brain cells. C_LIO_LIIn neuronal cells, {beta}-Amyloid oligomers target specific neuronal differentiation-related mRNAs to PBs. C_LIO_LIIn astroglia, {beta}-amyloid triggers the escape of inflammatory cytokine mRNAs from PBs. C_LIO_LIAltered mRNAs -protein phase separation connects PBs to neuroinflammation. C_LI

cell biology↗

bioRxiv2025

Change of Guards: ELAVL Proteins Switch miRNA Export Responsibility to Regulate Neuronal Differentiation

ELAVL1 protein HuR controls the stability and activity of miRNAs by disconnecting miRNPs from their target mRNAs and exporting Ago2-uncoupled miRNAs to help hepatic and macrophage cells manage stress. In mammalian neurons, the homolog of HuR, ELAVL4 or HuD, is expressed; however, its role in miRNA regulation remains unknown. PC12, a rat pheochromocytoma cell line, is a well-established model for studying neuronal differentiation that resembles sympathetic neurons. In PC12 cells, HuD levels increase during differentiation, which suppresses HuR expression. Thus, HuD overrides HuRs control of miRNA activity in differentiating cells, promoting differentiation by regulating the activity and levels of specific miRNAs, such as let-7a and miR-125b. When HuD is present, it decreases the activity of these miRNAs by binding to them, thereby facilitating their export and ensuring neuronal differentiation. Overall, there is a shift in miRNA regulation, with HuR being replaced by HuD in controlling miRNA export during neuronal differentiation. This switch in ELAVL-mediated miRNA regulation is essential for the differentiation process.

molecular biology↗

bioRxiv2025

HuR-Driven Reversible Mitochondrial Shuttling Buffers Cytosolic miRNA Levels in Hepatic Cells

Mitochondria, the "powerhouse" of mammalian cells, also serve as key storage sites for ions, metabolites, and enzymes vital for metabolic regulation. Exploring the regulatory processes that control the activities of miRNAs, the key non-coding RNA in mammalian cells, we found a context-dependent reversible localization of specific miRNAs to the mitochondrial matrix. Our data suggests a de novo role of mitochondria as miRNA sinks in mammalian cells. miR-122 is a key hepatic miRNA regulating metabolic processes in the mammalian liver. In this study, we observed increased mitochondrial targeting of miR-122 in amino acid-starved hepatic cells. Interestingly, when cells were refed with amino acids, mitochondrial miR-122 gets relocalized and reused in the cytosol for the translational repression process. Moreover, this phenomenon is not limited to miR-122 as several mitochondrial miRNAs (mito-miRs) follow similar transient storage inside mitochondria in stressed cells. Remarkably, mitochondria-localized mito-miRs preferentially target mRNAs encoding crucial mitochondrial components related to apoptosis. Hence, hepatic cells regulate apoptosis pathways during the starvation-refeeding cycle by shuttling a specific set of miRNAs to and from mitochondria, thereby balancing cytosolic miRNA content and homeostasis. Stress response miRNA binder ELAVL1 or HuR protein was found to be both necessary and sufficient for transporting the mature mito-miRs to the mitochondrial matrix - a process also controlled by the interaction between mitochondria and the endoplasmic reticulum. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/647748v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1178f5dorg.highwire.dtl.DTLVardef@1c5578borg.highwire.dtl.DTLVardef@f68555org.highwire.dtl.DTLVardef@ab9c40_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LISpecific miRNAs (mito-miRs), including miR-122, are targeted to mitochondria for transient storage in stressed hepatic cells. C_LIO_LIMito-miRs storage in mitochondria allows the expression of apoptosis-related genes in stressed cells. C_LIO_LIMito-miRs relocalization to cytoplasm on stress reversal allows suppression of apoptosis. C_LIO_LIBinding with ELAVL1 protein HuR allows reversible shuttling of mito-miRs to and from mitochondria in hepatic cells. C_LIO_LIER-mitochondrial interaction is key for the mitochondrial localization of miRNAs. C_LI

cell biology↗

bioRxiv2024

Amyloid Beta Oligomers Accelerate ATP-Dependent Phase Separation of Ago2 to RNA Processing Bodies

Phase separation to insoluble membrane-less organelles is a major way of activity regulation of specific proteins in eukaryotic cells. miRNA-repressed mRNAs and Ago proteins are known to be localized to RNA-processing bodies, the subcellular structures which are formed due to assembly of several RNA binding and regulatory proteins in eukaryotic cells. Ago2 is the most important miRNA binding protein that by forming complex with miRNA binds to mRNAs having cognate miRNA binding sites and represses protein synthesis in mammalian cells. Factors which control compartmentalization of Ago2 and miRNA-repressed mRNAs to RNA processing bodies are largely unknown. We have adopted a detergent permeabilized cell-based assay system to follow the phase separation of exogenously added Ago2 to RNA processing bodies in vitro. The Ago2 phase separation process is ATP dependent and is influenced by osmolarity and salt concentration of the reaction buffer. miRNA binding of Ago2 is essential for its targeting to RNA processing bodies and the compartmentalization process gets retarded by miRNA binding "sponge" protein HuR. This assay system found to be useful in identification of amyloid beta oligomers as miRNA-activity modulators which repress miRNA activity by enhancing Ago2-miRNP targeting to RNA processing bodies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=126 SRC="FIGDIR/small/584939v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1fe06eeorg.highwire.dtl.DTLVardef@7904b1org.highwire.dtl.DTLVardef@1231746org.highwire.dtl.DTLVardef@cd1405_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LImiRNA bound Ago2 gets phase separated in vitro to RNA processing bodies (PBs) in detergent permeabilized mammalian cells. C_LIO_LIPhase separation of Ago2 to PBs is controlled by presence of ATP and RNA. C_LIO_LIAmyloid beta oligomers retard dynamics of Ago2 bodies to inhibit miRNA function and enhance PB targeting of Ago2 miRNPs. C_LIO_LImicroRNA binding protein HuR can rescue Ago2 miRNP from PBs and inverse the effect of amyloid beta oligomers. C_LI

neuroscience↗

bioRxiv2023

RNA Binding of Syntaxin 5 Synergises Membrane Fusion to Accelerate miRNA Export and Clear Pathogens

Intercellular miRNA exchange acts as a key mechanism to control gene expression post-transcriptionally in mammalian cells. Regulated export of repressive miRNAs allows the expression of inflammatory cytokines in activated macrophages. Intracellular trafficking of miRNAs from endoplasmic reticulum to endosomes is a rate determining step in miRNA export process and plays an important role in controlling cellular miRNA level and inflammatory processes in macrophages. We have identified the SNARE protein Syntaxin5 to show a synchronized expression pattern with miRNA activity loss in activated mammalian macrophage cells. Syntaxin 5 is both necessary and sufficient for macrophage activation and clearance of the intracellular pathogen Leishmania donovani from infected macrophages. Exploring the mechanism of how Syntaxin5 acts as an immunostimulant, we have identified the de novo RNA binding property of this SNARE protein that binds specific miRNAs and facilitates their accumulation in endosomes in a cooperative manner with human ELAV protein HuR to ensure export of miRNAs and allows the expression of miRNA-repressed cytokines. Conversely, in its dual role in miRNA export, this SNARE protein prevents lysosomal targeting of endosomes by enhancing the fusion of miRNA-loaded endosomes with plasma membrane to ensure accelerated release of extracellular vesicles and associated miRNAs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=147 SRC="FIGDIR/small/556844v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@bfabf8org.highwire.dtl.DTLVardef@1b35346org.highwire.dtl.DTLVardef@203a5corg.highwire.dtl.DTLVardef@1d7a15e_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIHuR transfers miRNAs to STX5 for export C_LIO_LISTX5 utilizes its N-terminal disordered domain to bind and target miRNAs to endosomes. C_LIO_LIIn its dual role, STX5 uses the SNARE domain to export miRNA by promoting EV biogenesis and release. C_LIO_LISTX5-mediated miRNA export activates macrophage to clear internalized parasites. C_LI

immunology↗

bioRxiv2023

HuR-miRNA complex Activates RAS GTPase RalA to Facilitate Endosome Targeting and Extracellular Export of miRNAs

Extracellular vesicles-mediated exchange of miRNA cargos between diverse types of mammalian cells is a major mechanism of controlling cellular miRNA levels and activity and thus to regulate expression of miRNA-target genes in both donor and recipient cells. Despite tremendous excitement related to extracellular vesicles-associated miRNAs as biomarkers or having therapeutic potential, the mechanism of selective packaging of miRNAs into endosomes and multivesicular bodies for subsequent extracellular export is a poorly studied area due to lack of assay system to study such processes in vitro. We have developed an in vitro assay with endosomes isolated from mammalian macrophage cells to follow miRNA packaging into endocytic organelles. The synthetic miRNAs, used in the assay, get imported inside the isolated endosomes during the in vitro reaction and become protected from RNase in a time and concentration dependent manner. The selective miRNA accumulation inside endosomes requires both ATP and GTP hydrolysis and the miRNA binding protein HuR. The HuR-miRNA complex binds and stimulates the endosomal RalA GTPase to facilitate the import of miRNAs into endosomes and their subsequent export as part of the extracellular vesicles. The endosomal targeting of miRNAs is also very much dependent on endosome maturation process that is controlled by Rab5 protein and ATP. Graphical Abstract{whitebullet} miRNAs get imported to endosomes in vitro and are become protected to RNase {whitebullet}Endosomal miRNA import is sequence specific and requires miRNA exporter protein HuR {whitebullet}HuR-miRNA complex activates RalA GTPases to complete miRNA import. {whitebullet}Rab5 protein and ATP hydrolysis is required for endosome maturation and miRNA import O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/546187v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@217e52org.highwire.dtl.DTLVardef@ec6190org.highwire.dtl.DTLVardef@f972f5org.highwire.dtl.DTLVardef@ea67a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Leishmania Hijacks microRNA Import-Export Machinery of Infected Macrophage and Survives by Cross-Communicating Host miR-146a to Subjugate HuR and miR-122 in Neighbouring cells

Leishmania donovani, the causative agent of visceral leishmaniasis, infects and resides within tissue macrophage cells of the mammalian host. It is not clear how the parasite infected cells cross-talk with the non-infected cells in the infection niche to regulate the infection process. Interestingly, miRNAs, the regulatory small RNAs of the host, could get trafficked into and out of infected cells as part of extracellular vesicles to ensure exchange of the epigenetic signals and can regulate the expression of their target genes in both donor and recipient cells. Leishmania, for its survival in host macrophage, adopts a dual strategy to regulate the intercellular transport of host miRNAs. The parasite, by preventing mitochondrial function of the host cells, restricts the entry of liver cell derived miR-122 containing extracellular vesicles in infected macrophage to curtail the inflammatory response by miR-122. The parasite reciprocally upregulates the extracellular export of anti-inflammatory miR-146a from the infected cells. The exported miR-146a restricts miR-122 production in liver cells and polarizes neighbouring naive macrophages to the M2 state. miR-146a upregulates IL-10 in neighbouring macrophages where miR-146a dominates the RNA binding and miRNA suppressor protein HuR to inhibit the expression of proinflammatory cytokine mRNAs having HuR-interacting AU-rich elements and polarized the recipient cells to M2 stage. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/459146v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@32e32dorg.highwire.dtl.DTLVardef@e0d3c6org.highwire.dtl.DTLVardef@6f77e6org.highwire.dtl.DTLVardef@289e49_HPS_FORMAT_FIGEXP M_FIG C_FIG Key FindingsO_LILeishmania stops vesicular entry of inflammatory miR-122 in infected cells by causing mitochondrial depolarization C_LIO_LILeishmania secrets miR-146a from infected cells to stop miR-122 production in neighbouring hepatocytes C_LIO_LImiR-146a containing vesicles secreted by infected cells stops inflammatory response in recipient naive macrophage C_LIO_LImiR-146 targets RNA binding protein HuR to stop inflammatory cytokine production C_LI

immunology↗

bioRxiv2021

Target-Dependent Coordinated Biogenesis Ensure Cascaded Expression of miRNAs in Activated Macrophage

MicroRNAs (miRNAs) repress protein expression by binding to the 3 UTR of the target mRNAs. By exploring the effect of target mRNA on biogenesis of cognate miRNAs, we have noted miRNA with higher number of binding sites (primary miRNA) coordinates the biogenesis and activity of another miRNA (secondary miRNA) having binding sites on the 3 UTR of a common target mRNA. From the quantitative data obtained from macrophage cells, we detected miR-146a-5p as a "primary" miRNA that coordinates biogenesis of "secondary" miR-125b, miR-21 or miR-142-3p to target new sets of mRNAs to balance the immune response in activated macrophage cells. Interestingly, target dependent coordinated biogenesis of miRNAs, happening on the rough endoplasmic reticulum attached membrane, ensures a cumulative mode of action of primary and secondary miRNAs on the secondary target mRNAs where a cascaded effect of primary miRNA on its secondary targets has been detected. Extensive computational analysis for the presence of coordinated biogenesis pairs of miRNAs in mammalian cells has also allowed us to construct a coordinate biogenesis repository to determine context specific coordinated biogenesis relationships exists for specific pairs of miRNAs in mammalian cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/448041v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@318871org.highwire.dtl.DTLVardef@1f4d3a3org.highwire.dtl.DTLVardef@72adceorg.highwire.dtl.DTLVardef@f0d79e_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LImiRNA with higher number of binding sites (primary miRNA) can coordinates the biogenesis and activity of another miRNA (secondary miRNA) C_LIO_LITarget dependent coordinated biogenesis of miRNAs ensures a cumulative mode of action of primary and secondary miRNAs on the secondary target mRNAs on rER attached polysomes C_LIO_LImiR-146a-5p acts as a "primary" miRNA to coordinate biogenesis of "secondary" miR-125b, miR-21 or miR-142-3p in activated macrophage cells C_LIO_LICoordinate biogenesis balance the immune response in activated macrophage cells by ensuring propagation of primary miRNAs effect to diverse target mRNAs through secondary miRNAs. C_LI

molecular biology↗

bioRxiv2021

Rheb-mTOR Activation Rescues Amyloid Beta-Induced Cognitive Impairment and Memory Function by Restoring miR-146 Activity in Glial Cells

Deposition of amyloid beta plaques in adult rat or human brain is associated with increased production of proinflammatory cytokines by associated glial cells that are responsible for degeneration of the diseased tissue. The expression of these cytokines is usually under check and is controlled at post-transcriptional level via several microRNAs. Computational analysis of gene expression profiles of cortical regions of Alzheimers disease patients brain suggests ineffective target cytokine mRNA suppression by existing microRNPs in diseased brain. Exploring the mechanism of amyloid beta induced cytokine expression, we have identified how the inactivation of the repressive miR-146 microRNPs causes increased production of cytokines in amyloid beta exposed glial cells. In exploration of the cause of miRNP inactivation, we have noted amyloid beta oligomer induced sequestration of mTORC1 complex to early endosomes that results in decreased Ago2 phosphorylation, limited Ago2-miRNA uncoupling and retarded Ago2-cytokine mRNA interaction in rat astrocyte cells. Interestingly, constitutive activation of mTORC1 by Rheb activator restricts proinflammatory cytokine production by reactivating miR-146 microRNPs in amyloid beta exposed glial cells to rescue the disease phenotype in the in vivo rat model of Alzheimers disease.

neuroscience↗

bioRxiv2021

Inhibition of Extracellular Vesicle-Associated MMP2 Abrogates Intercellular Transfer of Hepatic miR-122 to Tissue Macrophages and Curtails Liver Inflammation

microRNA-122 (miR-122), a liver specific regulatory RNA, plays an important role in controlling metabolic homeostasis in mammalian liver cells. Interestingly, miR-122 is also a proinflammatory microRNA and when exported to tissue resident macrophage induces expression of inflammatory cytokines there. We found intercellular transfer of miR-122 in lipid exposed liver plays a role in liver inflammation. Exploring the mechanism of intercellular miR-122 transfer from hepatic cells, we detected MMP2 on the membrane of extracellular vesicles derived from hepatic cells which proved to be essential for transfer of extracellular vesicles and their miRNA content from hepatic to non-hepatic cells. Matrix metalloprotease 2 or MMP2 is a metalloproteinase that plays a key role in shaping and remodelling the extracellular matrix of human tissue by targeting degradation of matrix proteins. MMP2 was found to increase the movement of the EVs along the extracellular matrix to enhance their uptake in recipient cells. Inhibition of MMP2 restricts functional transfer of hepatic miRNAs across the hepatic and non-hepatic cell boundaries. By targeting MMP2, we could reduce the innate immune response in mammalian liver by preventing intra-tissue miR-122 transfer. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/425217v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@aec3b0org.highwire.dtl.DTLVardef@1b18e87org.highwire.dtl.DTLVardef@47e75org.highwire.dtl.DTLVardef@10a48e0_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIHuman hepatocytes on exposure to high lipid export out miRNAs including proinflammatory miR-122. C_LIO_LIExtracellular miR-122 is taken up by tissue macrophages to get them activated to produce inflammatory cytokines. C_LIO_LIMMP2 present on the surface of the EVs released by hepatocyte is essential for miRNA transfer to macrophage cells C_LIO_LIInhibition of MMP2 prevents miR-122 transfer to macrophage and stops activation of recipient macrophage. C_LI

cancer biology↗

bioRxiv2020

Amyloid Beta Oligomers Prevents Lysosomal Targeting of miRNP to Stop Its Recycling and Target Cytokine Repression in Glial Cells

mRNAs encoding inflammatory cytokines are targeted by miRNAs and remain repressed in neuroglial cells. On exposure to amyloid beta 1-42 oligomers, glial cells start expressing proinflammatory cytokines although there has been increase in repressive miRNAs levels as well. Exploring the mechanism of this potential immunity of target cytokine mRNAs against repressive miRNAs in amyloid beta exposed glial cells, we have identified differential compartmentalization of repressive miRNAs in glial cells to explain this aberrant miRNA function. While the target mRNAs were found to be associated with polysomes attached to endoplasmic reticulum, the miRNPs found to be present predominantly with endosomes that failed to recycle to endoplasmic reticulum attached polysomes to repress mRNA targets in treated cells. Amyloid beta oligomers, by masking the Rab7 proteins on endosomal surface, affects Rab7 interaction with Rab Interacting Lysosomal Protein (RILP) on lysosomes to restrict endosomal maturation and its lysosomal targeting. This causes retarded miRNP targeting to lysosomes and recycling. Similar defects in miRNP retrieval has been observed in endosome maturation defective cells depleted for RILP or treated with Bafilomycin. RNA processing body localization of the miRNPs was also noted in treated cells that happens as a consequence of enhanced endosomal retention of miRNPs. Interestingly, depletion of P-body partly rescues the miRNA function in glial cells exposed to amyloid beta and restricts the excess cytokine expression there. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/424324v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1d9453dorg.highwire.dtl.DTLVardef@150a9c4org.highwire.dtl.DTLVardef@b99a1aorg.highwire.dtl.DTLVardef@1342676_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LIAmyloid beta exposure causes accumulation of inactive miR-146 miRNP to cause elevated proinflammatory cytokine production in glial cells. C_LIO_LIAmyloid beta masks Rab7-RILP interaction to reduce endosome lysosome interaction. C_LIO_LIAccumulated miRNPs failed to get targeted to lysosomes in amyloid exposed cells due to loss of endosome lysosome interaction C_LIO_LILysosomal compartmentalization of miRNPs is required for its recycling and repression of de novo targets C_LIO_LIAccumulated miRNPs are stored in P-Bodies and depletion of P-Bodies rescues miRNA function in amyloid exposed glial cells. C_LI

cell biology↗

bioRxiv2020

Lipid Droplets Promote Phase Separation of Ago2 to Accelerate Dicer1 Loss and Decelerate miRNA Activity in Lipid Exposed Hepatic Cells

miR-122 is a liver specific miRNA that plays an important role in controlling metabolic homeostasis in mammalian liver cells. Interestingly, miR-122 on exposure to lipotoxic stress is reduced in liver cells. To fight stress, miRNA processor Dicer1 is depleted to cause reduced miR-122 production and the lowering of miRNA level ensures a better stress response in hepatocytes under lipotoxic stress. Interestingly, lipid droplets, formed in the liver cells on exposure to high fat, ensure cytoplasmic phase separation of Ago2 and prevent interaction of Ago2 with Dicer1. Lipid droplets bind miRNA and enhance miRNA-Ago2 uncoupling and Ago2 phase separation. Loss of interaction between Ago2 and Dicer1 eventually facilitates export and lowering of cellular Dicer1, a process also dependent on the endosomal maturation controller protein Alix, thereby ceasing pre-miRNA processing by Dicer1 in lipid exposed cells. Depletion of lipid droplets by downregulation of Perilipins with siRNAs resulted in a rescue of cellular Dicer1 level and Ago2-Dicer1 interaction. This is a novel mechanism that liver cells adopt to restrict cellular miRNA levels under stress condition. Thus, lipid droplets prevent cell death upon exposure to high fat by reducing intra and extracellular pool of miR-122 in hepatic tissue.

cell biology↗

bioRxiv2020

GW182 Proteins Restrict Extracellular Vesicle Mediated Selective Export of Used miRNAs In Mammalian Cancer Cells

MicroRNAs are small regulatory RNAs of relatively long half-life in non-proliferative human cells. However, in cancer cells the half-lives of miRNAs are comparatively short. To understand the mechanism of rapid miRNA turn over in cancer cells, we explored the effect of "usage" of specific miRNAs for translation repression of their targets on the abundance of that miRNA. We have noted an accelerated extracellular vesicle mediated export of "used" miRNAs in mammalian cells and the miRNA-export process get retarded by Ago2 interacting protein GW182B. The GW182 group of proteins are localized to GW182 Bodies or RNA Processing Bodies in mammalian cells and GW182B dependent retardation of miRNA export depends on GW-body integrity and is independent of HuR mediated auxiliary pathway of miRNA export. Our data thus support existence of a HuR independent pathway of miRNA export in human cells that can be targeted to increase cellular miRNA levels in cancer cells to induce senescence. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/294488v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1d6e30dorg.highwire.dtl.DTLVardef@d4ab06org.highwire.dtl.DTLVardef@282ad4org.highwire.dtl.DTLVardef@113ef76_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LIUsage of miRNA is proportional with its export and turnover C_LIO_LIGW182 proteins cause phase separation of Ago2 to P-bodies and restrict miRNA export via extracellular vesicles (EVs) C_LIO_LIHuR driven auxiliary path of miRNA export is independent of GW182 controlled main path of miRNA exclusion C_LI

molecular biology↗

bioRxiv2020

Non-Canonical Ago Loading of EV-Derived Exogenous miRNA Generates Foreign miRNPs on Endosomes to Arbitrate Gene Expression in Recipient Cells

MicroRNAs, the tiny regulators of gene expression, can be transferred between neighbouring cells via Extracellular Vesicles (EV) to control the expression of genes in both donor and recipient cells. How the Extracellular Vesicles-derived miRNAs are getting internalized and become functional in target cells is an unresolved question. We found that EV-associated miRNAs are primarily single stranded and, to become functional, get loaded with the Ago proteins present in the recipient cells without requiring host Dicer1. Following endocytosis of miRNA-containing extracellular vesicles, loading of host Ago occurs on the endosomal membrane where pH-dependent membrane fusion triggers the release of internalized miRNAs to form exogenous miRNP pool. In addition, loading of exogenous miRNA to host Ago also depends on the mitochondrial activity of recipient cells. Internalization of hepatocyte derived miR-122 containing EVs in macrophage activates the recipient cell by targeting cytokine expression. Leishmania donovani, a protozoan parasite known to affect endocytosis and to cause mitochondrial depolarization in infected macrophages, restricts the EV-internalization process and thereby preventing inflammatory cytokine expression and ensuring internalized pathogen survival in macrophage.

cell biology↗

bioRxiv2020

Mitochondria Controlled mTORC1 Activation Compartmentalizes Translation Initiation Factor eIF4E to Augment Intracellular Trafficking and Extracellular Export of miRNA in Mammalian Cells

Defective intracellular trafficking and export of miRNAs has been observed in senescent mammalian cells having impaired mitochondrial potential. Similar to what happens in senescent cells, Uncoupling Protein 2 mediated depolarization of mitochondrial membrane potential results in progressive sequestration of miRNAs with polysomes and lowered release of miRNAs through extracellular vesicles. Supporting importance of mitochondrial membrane potential on miRNAs fate determination, impaired miRNA-trafficking process in growth retarded human cells has been found to be reversed in presence of Genipin an inhibitor of Uncoupling Protein 2. Mitochondrial detethering of endoplasmic reticulum in mitochondria depolarized cells, found to be responsible for defective compartmentalization of translation initiation factor eIF4E to ER attached polysomes. It causes retarded translation process of target mRNAs with rER attached polysomes to ensure reduced intracellular trafficking and extracellular export of miRNAs. We have identified a reduced activity of mTORC1 complex in mitochondria defective cells to cause reduced phosphorylation of eIF4E-BP1 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 sub-cellular locations and export of miRNPs by affecting mTORC1 complex, the regulator of the protein translational machinery. Significance statementHow the reduced mitochondrial activity in growth retarded cells causes defective miRNA export is an open question. Mitochondrial defects induces a retarded subcellular miRNP trafficking in human cells to cause an upregulation in cellular miRNA content by reducing extracellular vesicle-mediated export of miRNA. We have identified a defective compartmentalization of translation initiation factor eIF4E in mitochondria-ER detethered mammalian cells to cause the retarded intracellular miRNA movement and export Activity of mTORC1 complex, a key regulator of protein translation in mammalian cells, is found to be responsible for ER-compartmentalization of eIF4E. mTORC1 activity reduction in growth retarded and mitochondria detethered cells influences the cell fate by acting on miRNA-mRNA axis. This is a unique way how mitochondrial activity is linked with protein translation and gene repression control in mammalian cells.

molecular biology↗

bioRxiv2018

The Constriction Ring of Amniotic Band Syndrome Reveals Idiosyncrasies of Wound Repair in Infancy

We hypothesized that the constriction rings in Amniotic Band Syndrome (ABS) are the sequelae of localized mechanical injury. Typical scarring pattern was examined in skin tissue of ABS origin, containing an amniotic band constriction, for collagen and elastin distribution, and the ratio of collagen I to collagen III (CI:CIII). A skin sample from an extra finger was the control. In the ABS specimens, sub-epidermal structures were intact and present throughout, and collagen I exhibited a normal basket-weave pattern. At the site of constriction in both ABS samples, reticular dermis elastin fibers were fragmented and papillary dermis elastin fibers were absent. In the control tissue, the reticular dermis contained relatively thick, branching fibers of elastin, and papillary dermis elastin was present.\n\nThe elastin fragmentation at the constriction ring indicates localized elastin remodeling in response to injury. The absence of elastin in the papillary dermis of the constriction likely indicates a localized disruption in elastin formation. The formation and deposition of collagen and the presence of sub-epidermal structures favor a non-scarring phenotype, while the organization of elastin favors that of localized scarring.\n\nSummary StatementA rare, fetal model of healing after constrictive injury of skin, in which elastin fragmentation at the constriction injury indicates remodeling explained by the differential expression of elastin during gestation.

pathology↗