Interaction Of miRNP Machinery With Subcellular Structures And Organelles: Implications In miRNA Mediated Gene Repression Process In Mammalian Cells

Chakrabarti, Yogaditya (2017) Interaction Of miRNP Machinery With Subcellular Structures And Organelles: Implications In miRNA Mediated Gene Repression Process In Mammalian Cells. PhD thesis, University of Calcutta.


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    Bhattacharyya, Suvendra Nath


    Any event inside the cell is determined by the synergistic functioning of required proteins in a given time and space. Such spatiotemporal regulation is important for cells, granting them ability to respond successfully to the plethora of changes happening in their microenvironment in order to survive. Genes encode proteins and proteins dictate cellular functions. Information flows from DNA to RNA to protein, according to the central dogma of molecular biology and each step is under stringent regulatory control to ensure cell fate and function. DNA or the cryptic code of life is first copied into small messages as messenger RNA (mRNA) molecules through transcription and then decryptified to the effector form as proteins by translation. Cells can control where, when and how much of a particular gene is transcribed and thereby translated. Regulation of these two major steps, transcription and translation is critical for the adaptability of the cell. Regulation of transcription and translation occurs in both prokaryotes and eukaryotes, but it is far more complex in eukaryotes. MicroRNA, the 22-24 nucleotides long non-coding RNA, forms miRNARibonucleoprotein (miRNP) complex with Argonaute (Ago) proteins and posttranscriptionally regulate diverse biological processes in metazoan cells. miRNA mediated gene repression process occurs via either translation inhibition or mRNA degradation (Bartel, 2009). Information on the subcellular sites where the miRNAmediated repression happens is still profoundly limited. Recently, several studies in plants as well as animals have established the association of miRNP complex with endomembranes during miRNA biogenesis and repressive function (Barman and Bhattacharyya, 2015; Kim et al., 2014). In particular, endoplasmic reticulum (ER) has been reported as the site for RISC (RNA induced silencing complex) nucleation as well as translational repression whereas, RISC accumulation and possible action is shown to be associated with endosomes and Multi-Vesicular Bodies (MVBs) (Gibbings et al., 2009; Lee et al., 2009; Li et al., 2013; Rogers and Chen, 2013; Stalder et al., 2013). So far none of the studies have linked miRNA based processes happening on ER and endosomal compartments and a lacuna still remains in understanding of ER and Endosome associated miRNA turnover. Other key organelles like mitochondria, primarily considered as power-houses in eukaryotic cells, show enrichment for a specific subset of miRNAs (Bandiera et al., 2011). Additionally, effect of mitochondrial membrane potential disruption on RNA interference and dynamics of cytoplasmic RNA granules such as Processing-body (P-body) have been documented in mammalian cells (Huang et al., 2011a; Kren et al., 2009). Cumulatively, these evidences indicate towards a spatial as well as temporal association of miRNPs with different organelles. But how these subcellular pools of miRNP complexes interact and exchange their components has not been addressed before. This phenomenon has potential role in modulation of miRNA activity and stability. miRNAs are relatively stable molecules with predominantly slow rate of turnover in animal cells (Carrion et al., 2013; Fabian et al., 2010; Gregory et al., 2008). For ensuring rapid switching between subcellular miRNA repertoire, enabling a single miRNA to repress multiple targets (Hutvagner and Zamore, 2002; Mourelatos et al., 2002), recycling of miRNP is essential. But, how these organellar pools of miRNAs, complexed with Ago proteins get replaced with new miRNAs to target different sets of genes is largely unknown. Specific changes in cellular miRNA profile under altered cellular environment have been reported. Many human diseases are associated with defective miRNA mediated repression of target mRNAs (Mendell and Olson, 2012). Multitudes of pathogenic parasites are known to alter the miRNA mediated repression process to their own advantage (Huang et al., 2011b; Manzano-Roman and Siles-Lucas, 2012; Skalsky and Cullen, 2010). Leishmania donovani (Ld), is a dimorphic pathogenic parasite which causes visceral leishmaniasis or kala-azar in vertebrates (Murray et al., 2005). Upon its entry through sand fly bite (Desjardins and Descoteaux, 1998; Engwerda et al., 2004; Olivier et al., 2005) for efficient infection of macrophages, Ld downregulates the pro-inflammatory response of host macrophages through induced expression of the Uncoupler Protein 2 (Ucp2) (Basu Ball et. al., 2011; Rousset et al., 2006), a mitochondrial membrane protein which modulates mitochondrial membrane potential by uncoupling the electron transport chain (Skulachev, 1998). In this study it has been reported that the pathogen Leishmania donovani invades the host macrophage and induces expression of Ucp2 and downregulates Mitofusin (Mfn) 2 to depolarize mitochondria and detether endosomes from ER causing defect in miRNP recycling. This miRNP recycling is essential to maintain the miRNA-AGO2 (miRISC) complex homeostatic levels by controlling the subcellular distribution of miRNA biogenesis associated factors and their interaction with each other. Moreover, this loss of functionality of mitochondrial membrane potential (ΔΨM) sensitive mitochondria-ER juxtapositioning also regulates P-body biogenesis by affecting the miRNA repression process. Furthermore, this phenomenon is also mimicked in high density culture based physiological paradigm. Mfn1 and Mfn2 are mitochondrial dynamin related GTPases. Global ablation of both Mfn1 and Mfn2 gene results in embryonic death. Remarkably, with conditional knockout crerecombinase system although the mice are born alive but, only the mice ubiquitously lacking MFN1 are apparently healthy, whereas mice without MFN2 die in the early postnatal period as well as show severe defects (Chen et al., 2003; Chen et al., 2007). Mfn2 with its lower GTPase activity is present on both mitochondrial and ER membranes and plays a key role in stabilizing ER-mitochondria tethering apart from its well-established role in mitochondrial fusion (Chang et al., 2004; de Brito and Scorrano, 2008; Ishihara et al., 2004; Schrepfer and Scorrano, 2016). Mfn2 ablation or depletion results in reduced colocalisation of ER with endosomes. Further experimentation with non-macrophage cells, allowed us to document reduced recycling of existing miRNP complexes and curtailed rate of miRNP biogenesis due to mitochondria-ER de-tethering. Thus, a unique mode of post-transcriptional gene regulation was identified in mammalian cells where inter-organellar interaction could control turnover and activity of cellular miRNA-Ago complex. This process is affected by Ld to alter miRNA activity during the infection of host macrophage cells that restricts production of pro-inflammatory cytokines.

    Item Type: Thesis (PhD)
    Subjects: Molecular & Human Genetics
    Divisions: UNSPECIFIED
    Depositing User: Dr N C Ghosh
    Date Deposited: 18 Jun 2018 15:13
    Last Modified: 18 Jun 2018 15:16

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