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

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Title Interaction Of miRNP Machinery With Subcellular
Structures And Organelles: Implications In miRNA
Mediated Gene Repression Process In Mammalian Cells
 
Creator Chakrabarti, Yogaditya
 
Subject Molecular & Human Genetics
 
Description 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.
 
Date 2017
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/2772/1/YOGADITYA_CHAKRABARTY_Ph.D._THESIS.pdf
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.
 
Relation http://www.eprints.iicb.res.in/2772/