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, thus regulating the 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 poorly studied due to the lack of an in vitro assay system. Here, 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 the endosome maturation process that is controlled by Rab5 protein and ATP. In summary, we provide an in vitro method to aid in the investigation of the mechanism of miRNA packaging process for its export from mammalian macrophage cells.

Human antigen R transfers miRNA to Syntaxin 5 to synergize miRNA export from activated macrophages.

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 the endoplasmic reticulum to endosomes is a rate-determining step in the miRNA export process and plays an important role in controlling cellular miRNA levels and inflammatory processes in macrophages. We have identified the SNARE protein Syntaxin 5 (STX5) to show a synchronized expression pattern with miRNA activity loss in activated mammalian macrophage cells. STX5 is both necessary and sufficient for macrophage activation and clearance of the intracellular pathogen Leishmania donovani from infected macrophages. Exploring the mechanism of how STX5 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 ELAVL1 protein, Human antigen R. This activity ensures the 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 the plasma membrane to ensure accelerated release of extracellular vesicles and associated miRNAs.

Accelerated export of Dicer1 from lipid-challenged hepatocytes buffers cellular miRNA-122 levels and prevents cell death.

Hepatocytes on exposure to high levels of lipids reorganize the metabolic program while fighting against the toxicity associated with elevated cellular lipids. The mechanism of this metabolic reorientation and stress management in lipid-challenged hepatocytes has not been well explored. We have noted the lowering of miR-122, a liver-specific miRNA, in the liver of mice fed with either a high-fat diet or a methionine-choline-deficient diet that is associated with increased fat accumulation in mice liver. Interestingly, low miR-122 levels are attributed to the enhanced extracellular export of miRNA processor enzyme Dicer1 from hepatocytes in the presence of high lipids. Export of Dicer1 can also account for the increased cellular levels of pre-miR-122-the substrate of Dicer1. Interestingly, restoration of Dicer1 levels in the mouse liver resulted in a strong inflammatory response and cell death in the presence of high lipids. Increasing death of hepatocytes was found to be caused by increased miR-122 levels in hepatocytes restored for Dicer1. Thus, the Dicer1 export by hepatocytes seems to be a key mechanism to combat lipotoxic stress by shunting out miR-122 from stressed hepatocytes. Finally, as part of this stress management, we determined that the Ago2-interacting pool of Dicer1, responsible for mature microribonucleoprotein formation in mammalian cells, gets depleted. miRNA-binder and exporter protein HuR is found to accelerate Ago2-Dicer1 uncoupling to ensure export of Dicer1 via extracellular vesicles in lipid-loaded hepatocytes.

Amyloid-ß oligomers block lysosomal targeting of miRNPs to prevent miRNP recycling and target repression in glial cells.

Upon exposure to amyloid-ß oligomers (Aß1-42), glial cells start expressing proinflammatory cytokines, despite an increase in levels of repressive microRNAs (miRNAs). Exploring the mechanism of this potential immunity of target cytokine mRNAs against repressive miRNAs in amyloid-ß-exposed glial cells, we have identified differential compartmentalization of repressive miRNAs in glial cells that explains this aberrant miRNA function. In Aß1-42-treated cells, whereas target mRNAs were found to be associated with polysomes attached to endoplasmic reticulum (ER), the miRNA ribonucleoprotein complexes (miRNPs) were found to be present predominantly with endosomes that failed to recycle to ER-attached polysomes, preventing repression of mRNA targets. Aß1-42 oligomers, by masking Rab7a proteins on endosomal surfaces, affected Rab7a interaction with Rab-interacting lysosomal protein (RILP), restricting the lysosomal targeting and recycling of miRNPs. RNA-processing body (P-body) localization of the miRNPs was found to be enhanced in amyloid-ß-treated cells as a consequence of enhanced endosomal retention of miRNPs. Interestingly, depletion of P-body components partly rescued the miRNA function in glial cells exposed to amyloid-ß and restricted the excess cytokine expression. This article has an associated First Person interview with the first author of the paper.

Non-canonical argonaute loading of extracellular vesicle-derived exogenous single-stranded miRNA in recipient cells.

MicroRNAs (miRNAs), the tiny regulators of gene expression, can be transferred between neighbouring cells via extracellular vesicles (EVs) to control the expression of genes in both donor and recipient cells. How the EV-derived miRNAs are internalized and become functional in target cells is an unresolved question. We have expressed a liver-specific miRNA, miR-122, in non-hepatic cells for packaging in released EVs. With these EVs, we have followed the trafficking of miR-122 to recipient HeLa cells that otherwise do not express this miRNA. We found that EV-associated miR-122 is primarily single-stranded and, to become functional, is loaded onto the recipient cell argonaute proteins without requiring host Dicer1. Following endocytosis, EV-associated miR-122 is loaded onto the host cell argonaute proteins on the endosomal membrane, where the release of internalized miRNAs occurs in a pH-dependent manner, facilitating the formation of the exogenous miRNP pool in the recipient cells. Endosome maturation defects affect EV-mediated entry of exogeneous miRNAs in mammalian cells. This article has an associated First Person interview with the first author of the paper.

Mitochondria control mTORC1 activity-linked compartmentalization of eIF4E to regulate extracellular export of microRNAs.

Defective intracellular trafficking and export of microRNAs (miRNAs) have been observed in growth-retarded mammalian cells having impaired mitochondrial potential and dynamics. Here, we found that uncoupling protein 2 (Ucp2)-mediated depolarization of mitochondrial membrane also results in progressive sequestration of miRNAs within polysomes and lowers their release via extracellular vesicles. Interestingly, the impaired miRNA-trafficking process in growth-retarded human cells could be reversed in the presence of Genipin, an inhibitor of Ucp2. Mitochondrial detethering of endoplasmic reticulum (ER), observed in cells with depolarized mitochondria, was found to be responsible for defective compartmentalization of translation initiation factor eIF4E to polysomes attached to ER. This caused a retarded translation process accompanied by enhanced retention of miRNAs and target mRNAs within ER-attached polysomes to restrict extracellular export of miRNAs. Reduced compartment-specific activity of the mammalian target of rapamycin complex 1 (mTORC1), the master regulator of protein synthesis, in cells with defective mitochondria or detethered ER, caused reduced phosphorylation of eIF4E-BP1 and prevented eIF4E targeting to ER-attached polysomes and miRNA export. These data suggest how mitochondrial membrane potential and dynamics, by affecting mTORC1 activity and compartmentalization, determine the subcellular localization and export of miRNAs.

Probing the molecular mechanism of aggressive infection by antimony resistant Leishmania donovani.

The disease visceral leishmaniasis (VL) or kala azar is caused by the protozoan parasite, Leishmania donovani (LD). For many decades the pentavalent antimonial drugs countered the successive epidemics of the disease in the Indian sub-continent and elsewhere. With time, antimony resistant LD (LDR) developed and the drug in turn lost its efficacy. Infection of mammals with LDR gives rise to aggressive infection as compared to its sensitive counterpart (LDS) coupled with higher surge of IL-10 and TGF-ß. The IL-10 causes upregulation of multidrug resistant protein-1 which causes efflux of antimonials from LDR infected cells. This is believed to be a key mechanism of antimony resistance. MicroRNAs (miRNAs) are tiny post-transcriptional regulators of gene expression in mammalian cells and in macrophage play a pivotal role in controlling the expression of cytokines involved in infection process. Therefore, a change in miRNA profiles of macrophages infected with LDS or LDR could explain the differential cytokine response observed. Interestingly, the outcome of LD infection is also governed by the critical balance of pro- and anti-inflammatory cytokines which is inturn regulated by miRNA-Ago2 or miRNP complex and its antagonist RNA binding protein HuR. Here Ago2 plays the fulcrum whose phosphorylation and de-phosphorylation dictates the process; which in turn is controlled by PP2A and HuR. LDS and LDR upregulate PP2A and downregulate HuR at different magnitude leading to various levels of anti-inflammatory to proinflammatory cytokine production and resulting pathology in the host. While ectopic HuR expression alone is sufficient to clear LDS infection, simultaneous upregulation of HuR and inhibition of PP2A is required to inhibit LDR mediated infection. Therefore, tampering with miRNA pathway could be a new strategy to control infection caused by LDR parasite.

Reversible HuR-microRNA binding controls extracellular export of miR-122 and augments stress response.

microRNAs (miRNAs), the tiny but stable regulatory RNAs in metazoan cells, can undergo selective turnover in presence of specific internal and external cues to control cellular response against the changing environment. We have observed reduction in cellular miR-122 content, due to their accelerated extracellular export in human hepatic cells starved for small metabolites including amino acids. In this context, a new role of human ELAV protein HuR has been identified. HuR, a negative regulator of miRNA function, accelerates extracellular vesicle (EV)-mediated export of miRNAs in human cells. In stressed cells, HuR replaces miRNPs from target messages and is both necessary and sufficient for the extracellular export of corresponding miRNAs. HuR could reversibly bind miRNAs to replace them from Ago2 and subsequently itself gets freed from bound miRNAs upon ubiquitination. The ubiquitinated form of HuR is predominantly associated with multivesicular bodies (MVB) where HuR-unbound miRNAs also reside. These MVB-associated pool of miRNAs get exported out via EVs thereby delimiting cellular miR-122 level during starvation. Therefore, by modulating extracellular export of miR-122, HuR could control stress response in starved human hepatic cells.

mRNA Targeting to Endoplasmic Reticulum Precedes Ago Protein Interaction and MicroRNA (miRNA)-mediated Translation Repression in Mammalian Cells.

MicroRNA (miRNA) binds to the 3'-UTR of its target mRNAs to repress protein synthesis. Extensive research was done to understand the mechanism of miRNA-mediated repression in animal cells. Considering the progress in understanding the mechanism, information about the subcellular sites of miRNA-mediated repression is surprisingly limited. In this study, using an inducible expression system for an miRNA target message, we have delineated how a target mRNA passes through polysome association and Ago2 interaction steps on rough endoplasmic reticulum (ER) before the miRNA-mediated repression sets in. From this study, de novo formed target mRNA localization to the ER-bound polysomes manifested as the earliest event, which is followed by Ago2 micro-ribonucleoprotein binding, and translation repression of target message. Compartmentalization of this process to rough ER membrane ensures enrichment of miRNA-targeted messages and micro-ribonucleoprotein components on ER upon reaching a steady state.

Insulin-like growth factor-1 prevents miR-122 production in neighbouring cells to curtail its intercellular transfer to ensure proliferation of human hepatoma cells.

miRNAs are 20-22 nt long post-transcriptional regulators in metazoan cells that repress protein expression from their target mRNAs. These tiny regulatory RNAs follow tissue and cell-type specific expression pattern, aberrations of which are associated with various diseases. miR-122 is a liver-specific anti-proliferative miRNA that, we found, can be transferred via exosomes between human hepatoma cells, Huh7 and HepG2, grown in co-culture. Exosomal miR-122, expressed and released by Huh7 cells and taken by miR-122 deficient HepG2 cells, was found to be effective in repression of target mRNAs and to reduce growth and proliferation of recipient HepG2 cells. Interestingly, in a reciprocal process, HepG2 secretes Insulin-like Growth Factor 1 (IGF1) that decreases miR-122 expression in Huh7 cells. Our observations suggest existence of a reciprocal interaction between two different hepatic cells with distinct miR-122 expression profiles. This interaction is mediated via intercellular exosome-mediated miR-122 transfer and countered by a reciprocal IGF1-dependent anti-miR-122 signal. According to our data, human hepatoma cells use IGF1 to prevent intercellular exosomal transfer of miR-122 to ensure its own proliferation by preventing expression of growth retarding miR-122 in neighbouring cells.

A transient reversal of miRNA-mediated repression controls macrophage activation.

In mammalian macrophages, the expression of a number of cytokines is regulated by miRNAs. Upon macrophage activation, proinflammatory cytokine mRNAs are translated, although the expression of miRNAs targeting these mRNAs remains largely unaltered. We show that there is a transient reversal of miRNA-mediated repression during the early phase of the inflammatory response in macrophages, which leads to the protection of cytokine mRNAs from miRNA-mediated repression. This derepression occurs through Ago2 phosphorylation, which results in its impaired binding to miRNAs and to the corresponding target mRNAs. Macrophages expressing a mutant, non-phosphorylatable AGO2--which remains bound to miRNAs during macrophage activation--have a weakened inflammatory response and fail to prevent parasite invasion. These findings highlight the relevance of the transient relief of miRNA repression for macrophage function.

Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight?

MicroRNAs constitute a large family of small, approximately 21-nucleotide-long, non-coding RNAs that have emerged as key post-transcriptional regulators of gene expression in metazoans and plants. In mammals, microRNAs are predicted to control the activity of approximately 30% of all protein-coding genes, and have been shown to participate in the regulation of almost every cellular process investigated so far. By base pairing to mRNAs, microRNAs mediate translational repression or mRNA degradation. This Review summarizes the current understanding of the mechanistic aspects of microRNA-induced repression of translation and discusses some of the controversies regarding different modes of microRNA function.

HuR protein attenuates miRNA-mediated repression by promoting miRISC dissociation from the target RNA.

The microRNA (miRNA)-mediated repression of protein synthesis in mammalian cells is a reversible process. Target mRNAs with regulatory AU-rich elements (AREs) in their 3'-untranslated regions (3'-UTR) can be relieved of miRNA repression under cellular stress in a process involving the embryonic lethal and altered vision family ARE-binding protein HuR. The HuR-mediated derepression occurred even when AREs were positioned at a considerable distance from the miRNA sites raising questions about the mechanism of HuR action. Here, we show that the relief of miRNA-mediated repression involving HuR can be recapitulated in different in vitro systems in the absence of stress, indicating that HuR alone is sufficient to relieve the miRNA repression upon binding to RNA ARE. Using in vitro assays with purified miRISC and recombinant HuR and its mutants, we show that HuR, likely by its property to oligomerize along RNA, leads to the dissociation of miRISC from target RNA even when miRISC and HuR binding sites are positioned at a distance. Further, we demonstrate that HuR association with AREs can also inhibit miRNA-mediated deadenylation of mRNA in the Krebs-2 ascites extract, in a manner likewise depending on the potential of HuR to oligomerize.

Target-Dependent Coordinated Biogenesis of Secondary MicroRNAs by miR-146a Balances Macrophage Activation Processes.

MicroRNAs (miRNAs) repress protein expression by binding to the target mRNAs. Exploring whether the expression of one miRNA can regulate the abundance and activity of other miRNAs, we noted the coordinated biogenesis of miRNAs in activated macrophages. miRNAs with higher numbers of binding sites (the "primary" miRNAs) induce expression of other miRNAs ("secondary" miRNAs) having binding sites on the 3' untranslated region (UTR) of common target mRNAs. miR-146a-5p, in activated macrophages, acts 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 responses. During coordinated biogenesis, primary miRNA drives the biogenesis of secondary miRNA in a target mRNA- and Dicer1 activity-dependent manner. The coordinated biogenesis of miRNAs was observed across different cell types. The target-dependent coordinated miRNA biogenesis also ensures a cumulative mode of action of primary and secondary miRNAs on the secondary target mRNAs. Interestingly, using the "primary" miR-146a-5p-specific inhibitor, we could inhibit the target-dependent biogenesis of secondary miRNAs that can stop the miRNA-mediated buffering of cytokine expression and inflammatory response occurring in activated macrophages. Computational analysis suggests the prevalence of coordinated biogenesis of miRNAs also in other contexts in human and in mouse.

Leishmania survives by exporting miR-146a from infected to resident cells to subjugate inflammation.

Leishmania donovani, the causative agent of visceral leishmaniasis, infects and resides within tissue macrophage cells. It is not clear how the parasite infected cells crosstalk with the noninfected cells to regulate the infection process. During infection, Leishmania adopts a dual strategy for its survival by regulating the intercellular transport of host miRNAs to restrict inflammation. The parasite, by preventing mitochondrial function of host cells, restricts the entry of liver cell derived miR-122-containing extracellular vesicles in infected macrophages to curtail the inflammatory response associated with miR-122 entry. On contrary, the parasite up-regulates the export of miR-146a from the infected macrophages. The miR-146a, associated with the extracellular vesicles released by infected cells, restricts miR-122 production in hepatocytes while polarizing neighbouring naïve macrophages to the M2 state by affecting the cytokine expression. On entering the recipient macrophages, miR-146a dominates the miRNA antagonist RNA-binding protein HuR to inhibit the expression of proinflammatory cytokine mRNAs having HuR-interacting AU-rich elements whereas up-regulates anti-inflammatory IL-10 by exporting the miR-21 to polarize the recipient cells to M2 stage.

GW182 Proteins Restrict Extracellular Vesicle-Mediated Export of MicroRNAs in Mammalian Cancer Cells.

MicroRNAs (miRNAs) 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 turnover in cancer cells, we explored the effect of target mRNAs on the abundance of the miRNAs that repress them. We have noted an accelerated extracellular vesicle (EV)-mediated export of miRNAs in presence of their target mRNAs in mammalian cells, and this target-driven miRNA-export process is 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 the HuR protein-mediated auxiliary pathway of miRNA export. Our data thus support the existence of a HuR-independent pathway of miRNA export in human cells that can be targeted in MDA-MB-231 cancer cells, to increase the level of cellular let-7a, a known negative regulator of cancer growth.

Rheb-mTOR activation rescues Aß-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 the post-transcriptional level via several microRNAs. Computational analysis of gene expression profiles of cortical regions of Alzheimer's disease patients' brain suggests ineffective target cytokine mRNA suppression by existing micro-ribonucleoproteins (miRNPs) in diseased brain. Exploring the mechanism of amyloid beta-induced cytokine expression, we have identified how the inactivation of the repressive miR-146 miRNPs 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 the mTORC1 complex to early endosomes that results in decreased Ago2 phosphorylation, limited Ago2-miRNA uncoupling, and retarded Ago2-cytokine mRNA interaction in rat astrocytes. Interestingly, constitutive activation of mTORC1 by Rheb activator restricts proinflammatory cytokine production by reactivating miR-146 miRNPs in amyloid beta-exposed glial cells to rescue the disease phenotype in the in vivo rat model of Alzheimer's disease.

Inhibition of extracellular vesicle-associated MMP2 abrogates intercellular hepatic miR-122 transfer to liver macrophages and curtails inflammation.

Hepatic miRNA, miR-122, plays an important role in controlling metabolic homeostasis in mammalian liver. Intercellular transfer of miR-122 was found to play a role in controlling tissue inflammation. miR-122, as part of extracellular vesicles released by lipid-exposed hepatic cells, are taken up by tissue macrophages to activate them and produce inflammatory cytokines. Matrix metalloprotease 2 or MMP2 was found to be essential for transfer of extracellular vesicles and their miRNA content from hepatic to non-hepatic cells. MMP2 was found to increase the movement of the extracellular vesicles 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, and by targeting MMP2, we could reduce the innate immune response in mammalian liver by preventing intra-tissue miR-122 transfer. MMP2 thus could be a useful target to restrict high-fat-diet-induced obesity-related metaflammation.

Retrograde trafficking of Argonaute 2 acts as a rate-limiting step for de novo miRNP formation on endoplasmic reticulum-attached polysomes in mammalian cells.

microRNAs are short regulatory RNAs in metazoan cells. Regulation of miRNA activity and abundance is evident in human cells where availability of target messages can influence miRNA biogenesis by augmenting the Dicer1-dependent processing of precursors to mature microRNAs. Requirement of subcellular compartmentalization of Ago2, the key component of miRNA repression machineries, for the controlled biogenesis of miRNPs is reported here. The process predominantly happens on the polysomes attached with the endoplasmic reticulum for which the subcellular Ago2 trafficking is found to be essential. Mitochondrial tethering of endoplasmic reticulum and its interaction with endosomes controls Ago2 availability. In cells with depolarized mitochondria, miRNA biogenesis gets impaired, which results in lowering of de novo-formed mature miRNA levels and accumulation of miRNA-free Ago2 on endosomes that fails to interact with Dicer1 and to traffic back to endoplasmic reticulum for de novo miRNA loading. Thus, mitochondria by sensing the cellular context regulates Ago2 trafficking at the subcellular level, which acts as a rate-limiting step in miRNA biogenesis process in mammalian cells.

MicroRNA exporter HuR clears the internalized pathogens by promoting pro-inflammatory response in infected macrophages.

HuR is a miRNA derepressor protein that can act as miRNA sponge for specific miRNAs to negate their action on target mRNAs. Here we have identified how HuR, by inducing extracellular vesicles-mediated export of miRNAs, ensures robust derepression of miRNA-repressed cytokines essential for strong pro-inflammatory response in activated mammalian macrophages. Leishmania donovani, the causative agent of visceral leishmaniasis, on the contrary alters immune response of the host macrophage by a variety of complex mechanisms to promote anti-inflammatory response essential for the survival of the parasite. We have found that during Leishmania infection, the pathogen targets HuR to promote onset of anti-inflammatory response in mammalian macrophages. In infected macrophages, Leishmania also upregulate protein phosphatase 2A that acts on Ago2 protein to keep it in dephosphorylated and miRNA-associated form. This causes robust repression of the miRNA-targeted pro-inflammatory cytokines to establish an anti-inflammatory response in infected macrophages. HuR has an inhibitory effect on protein phosphatase 2A expression, and mathematical modelling of macrophage activation process supports antagonistic miRNA-modulatory roles of HuR and protein phosphatase 2A which mutually balances immune response in macrophage by targeting miRNA function. Supporting this model, ectopic expression of the protein HuR and simultaneous inhibition of protein phosphatase 2A induce strong pro-inflammatory response in the host macrophage to prevent the virulent antimonial drug-sensitive or drug-resistant form of L. donovani infection. Thus, HuR can act as a balancing factor of immune responses to curtail the macrophage infection process by the protozoan parasite.