BHATTACHARYYA LAB
RNA Biology Research Laboratory
Compartmentalization of miRNA-mediated gene repression machineries in mammalian cells
Cytoplasm of eukaryotic cells is subdivided into membrane bound compartments called organelles. Intracellular organelles not only provide membrane-enclosed compartments separated from the cytosol but also create increased membrane surfaces. Most of the vital biochemical processes of the cell take place on membrane surfaces. Compartmentalization of cellular processes to specific subcellular addresses is a mechanism that ensures stringent regulation of biochemical reactions and processes. Compartmentalization of transcription to specific nuclear territories or compartmentalized translation on rough endoplasmic reticulum (rER) membrane serves to regulate these processes with exquisite spatial and temporal control.
Ago2, miRNAs and target mRNAs have all been found to localize to processing bodies (P-bodies), the cytoplasmic sites for mRNA degradation. However, it has been observed that visible P-bodies are not required for RNAi and their formation is a consequence rather than a cause of silencing. Hence, the exact subcellular address of miRNA function has remained obscure despite ample conceptual advancement in understanding the mechanisms of miRNA-mediated gene regulation. A couple of evidences indicate a possible role of membranous structures in miRNA function. miRNAs and their target mRNAs are observed to enrich on the surface of rER membranes. More specifically, a newly synthesized mRNA localizes to rER attached polysomes which is followed by its binding with miRNP and translation repression (Barman and Bhattacharyya, 2015, J Biol. Chem.).
Translation repression by miRNA is followed by mRNA degradation. These two events are separated by time and space. This is achieved by compartmentalization of these two processes in distinct subcellular addresses. Repressed messages from rER are targeted to Multivesicular bodies (MVBs) where they are deadenylated and subsequently degraded.
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Regulation of miRNA transfer between mammalian cells
Cells release different types of vesicles into the extracellular milieu. Depending upon the mode of origin, these extracellular vesicles or EVs are classified either as microvesicles, ectosomes, shedding vesicles or exosomes. Exosomes are 40-100nm sized vesicles which are released by evagination of multivesicular bodies into the exterior. MicroRNAs have been found to circulate in body fluids either in association with the Argonaute protein or enclosed in exosomes. Recently it has been found that exosomes can serve as important mediators of cell to cell communication as they can transfer mRNAs, microRNAs among other cargo proteins and molecules which can remain functional in the recipient cells. This mode of communication has an important implication in the background of cancer and immunity.
However despite recent advances in exosomal microRNA biology, the mechanism of transfer of exosomal miRNA between mammalian cells is unclear. What are the factors which regulate the transfer of functional microRNAs via exosomes is also an avenue for study. We have observed that there exists a reciprocal regulation in controlling miRNAs transfer between hepatic cells. It was observed that exosomal miR-122 released by Huh7 cells is taken up by HepG2 cells causing retarded growth while the HepG2 cells in turn secrete Insulin-like Growth Factor 1 (IGF1) to decrease miR-122 expression in Huh7 cells to ensure its own proliferation (Basu et al., 2014, Nucleic Acids Res.).
There are several factors which regulate the loading of miRNAs into the exosomes. We have observed that during amino acid starvation in hepatic cells, there occurs a decrease in the cellular miR-122 content, due to their extracellular export in human hepatic cells facilitated by the human ELAV protein HuR. HuR was found to reversibly bind miRNAs to replace them from Ago2 and subsequently itself gets freed from bound miRNAs upon ubiquitination. Interestingly, by modulating extracellular export of miR-122, HuR could control stress response in starved human hepatic cells (Mukherjee et al. 2016, EMBO Rep.).
We are also interested in understanding the mechanism of transfer of miRNAs between mammalian cell, the pathways involved and the factors responsible for maintaining the functionality of the transferred miRNA in the recipient cells.
Modulation of miRNA binding of Ago proteins by intrinsic and extrinsic factors in neuronal and immune cells
Ago2 (Argonaute) is a major effector protein that causes miRNA mediated mRNA repression. Previous studies showed post translational modifications such as phosphorylation can alter Ago2 localization or small RNA binding thus affecting the gene expression profile.
In one of our previous study we elucidated a mechanism in which uncoupling of the miRNA let-7a from Ago2 occurs due to p-MSK1 mediated Ago2 phosphorylation in differentiating neuronal cells. A drop in the Let-7a activity ensures upregulation of its target KRas, essential for neuronal differentiation. We found inactivation of let-7a microRNP is both necessary and sufficient to cause differentiation in sympathetic neurons (Patranabis et al,2016, Mol Cell Biol).
In another study, we have reported how Ago2 phosphorylation and consequent incativation of miRNPs fine tunes the balance between pro-inflammatory and anti-inflammatory cytokines expression in mammalian macrophage. In an early phase of response to bacterial lipopolysaccarides, miRNAs dissociates from Ago2 which gets phosphorylated at its small RNA binding pocket in presence of pro-inflammatory signals. However this dissociation results in derepression of mRNAs targeted by miRNAs causing a transient surge of pro-inflammatory cytokine expression. During inflammatory response, these modifications in miRNA activity minutely controlled and reverted back to normal to avoid hyper-responsiveness and septic shock (Mazumder et al, 2013, EMBO Rep.).
Pathogen mediated alteration in miRNA-machinery in Leishmania invaded macrophage and neighboring non-macrophage cells
Leishmania donovani (Ld) is an intracellular parasite which has cleaver strategies to reside within the macrophages within a vesicle. However apart from macrophages, Ld also targets hepatic cells and modulate their metabolic genes. Research from our lab suggest modulation of miRNA machineries in Ld infected or interacting cells. Leishmania surface glycoprotein gp63, which happens to be a zinc metallo-protease cleavespre-miRNA processor Dicer1 to downregulate miR-122in hepatic cells interacting the pathogen. By reducing miR-122, Ld lowers serum cholesterol level in infected animals which facilitates establishment of the infection (Ghosh et al, 2013, Cell Host& Microbe).
In infected macrophage Ld upregulates Uncoupler Protein 2 (UCP2) which causes depolarization of mitochondrial membrane potential. Mitochondial abnormalities in infected macrophage causes reduced turnover of miRNPs in infected cells possibly by sequestered and rendered them inactive in specific subcellular compartment.
Ld infection even affects the recycling of miRNPS by blocking endosome maturation which directly increases the stability of several miRNA-target mRNAs. It is fascinating to understand the mechanism of these effects. A major research aim of RBRL is therefore to understand how the pathogen cleverly targets the host miRNA machinery to establish a suitable niche for its sustenance in infected cells.
We also decipher that by targeting hur and upregulation of pp2A, Ld modulates miRNA mediated repression of cytokines to establish anti- inflammatory response within the host cell that favor the progression of infection
