The SARS-CoV-2 protein NSP2 enhances microRNA-mediated translational repression.

Viruses use microRNAs (miRNAs) to impair the host antiviral response and facilitate viral infection by expressing their own miRNAs or co-opting cellular miRNAs. miRNAs inhibit translation initiation of their target mRNAs by recruiting the GIGYF2-4EHP (or EIF4E2) translation repressor complex to the mRNA 5'-cap structure. We recently reported that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-encoded non-structural protein 2 (NSP2) interacts with GIGYF2. This interaction is critical for blocking translation of the Ifnb1 mRNA that encodes the cytokine interferon ß, and thereby impairs the host antiviral response. However, it is not known whether NSP2 also affects miRNA-mediated silencing. Here, we demonstrate the pervasive augmentation of miRNA-mediated translational repression of cellular mRNAs by NSP2. We show that NSP2 interacts with argonaute 2 (AGO2), the core component of the miRNA-induced silencing complex (miRISC), via GIGYF2 and enhances the translational repression mediated by natural miRNA-binding sites in the 3' untranslated region of cellular mRNAs. Our data reveal an additional layer of the complex mechanism by which SARS-CoV-2 and likely other coronaviruses manipulate the host gene expression program by co-opting the host miRNA-mediated silencing machinery.

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.

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.

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.

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.

Intraocular pressure following combined routes of bevacizumab-augmented trabeculectomy for refractory neovascular glaucoma.

PURPOSE: To evaluate the intraocular pressure (IOP) control following combined routes of adjuvant bevacizumab with trabeculectomy in refractory neovascular glaucoma. METHODS: From June 2011 to December 2011, 5 consecutive cases of neovascular glaucoma with persistent raised IOP on maximal medical treatment underwent adjuvant bevacizumab by combined routes (subconjunctival (SC) and/or intracameral (IC), intravitreal (IV) injections) before pan-retinal photocoagulation (PRP). Needs for repeat procedures or medications for IOP control over the postoperative period were assessed. RESULTS: The mean IOP (1 SC, 1 IC + IV, 3 SC + IC routes) reduced from 40 ± 5.5 mm Hg to 17 ± 3.7 mm Hg at a mean final follow-up of 4 ± 3.7 months (range 1-9 months), respectively (p < 0.001 for each). All eyes had transient IOP spikes 1-3 months after surgery, which normalized spontaneously after PRP, while one eye required topical medications for IOP control. CONCLUSIONS: Combined routes of adjuvant bevacizumab augmented trabeculectomy may help in better IOP control (IC + IV > IC + SC > SC) in refractory neovascular glaucoma but require additional procedures for sustained effect.

20G silicone rod as monocanalicular stent in repair of canalicular lacerations: experience from a tertiary eye care centre.

To evaluate the outcome of 20G silicone rod as monocanalicular stent in canalicular lacerations. Retrospective case series involving patients between July 2006 and June 2010. Fourteen canalicular repairs in 12 consecutive patients were done in the study period. Eleven were male and mean age was 30.5 years. A single canaliculus was involved in 10 patients and associated injury to the globe was noted in 3 patients. The median lag time between injury and repair was 3 (range 1-9) days. The mean duration of stenting was 6.9 (SD 3.2) weeks. Spontaneous extrusion of monocanalicular stent occurred in 3 patients. Patency on syringing was noted in 10 (70%) canaliculi over a median follow up of 7 (range 2-17) months. 20G silicone rod may be used as an effective and economical alternative in canalicular lacration repairs.