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Role of Cellular Membranes in Controlling Micro RNA Mediated Co-ordinated Gene Expression

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Title Role of Cellular Membranes in Controlling Micro
RNA Mediated Co-ordinated Gene Expression
 
Creator CHATTERJEE, SUSANTA
 
Subject Molecular & Human Genetics
 
Description MicroRNAs are ~22 nt. regulatory RNAs that bind to 3’ UTR of target mRNA and
regulate the expression by translational repression or transcriptional inactivation. Previously
it has been reported there exists a target mRNA dependent miRNA biogenesis where target
mRNA could regulate its cognate miRNA biogenesis by modulating Dicer processivity. It is
well known that multiple miRNAs share the common 3’ UTR of the same target mRNA. So I
was curious to check the additive level of regulation in “target mRNA dependent miRNA
biogenesis” on other miRNAs that share binding sites on the same 3`UTR of target mRNA.
My aim was to dissect the molecular manifestation of this “cooperative regulation” and its
relevance in normal cellular physiology. Earlier It was observed there exists a target mRNA
dependent miRNA biogenesis where one target mRNA could regulate its cognate miRNA
biogenesis by modulating Dicer processivity. I was curious how this phenomenon affects
biogenesis of other miRNAs on the same 3`UTR. I could observe miRNA family with a
higher number of binding sites (miRNA 1) influences the biogenesis of adjacent groups of
miRNAs (miRNA 2) that shares the common 3’ UTR. Along with, I could also observe
repression of secondary mRNAs (mRNA B) that bear sites for miRNA2 (but not for
miRNA1) is co-ordinately regulated with miRNA1 abundance inside the cells.
Using a reporter based model in endotoxin-stress induced TLR4-activation module,
corroborate my findings to propose “target dependent cooperative biogenesis of miRNAs' '
phenomenon inside the mammalian cells. A computational module was developed to predict
regulatory miRNA and their network under different physiological contexts. Interestingly
again, many of those predicted cooperative miRNAs and its’ secondary target has been
revalidated biochemically that exhibits the same trend, strengthening the proposed
hypothesis. I could observe coordinated regulation of miRNAs resulting in alleviating
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bacterial endotoxin-induced pro-inflammatory response. My observations suggest "target
dependent cooperative biogenesis of miRNAs (TDCB)” added an additional layer of finetuning of signalling molecules on endotoxin-responsive murine macrophages to re-establish
cellular homeostasis. Additionally I have also shown mechanistically co-operative biogenesis
of miRNAs on macrophages play an important role to combat pro-inflammatory response.
Along with I have also explored the cellular compartmentalization of this phenomenon.
Previous reports suggest polysome attached with rER serves as the nucleation site for miRNP
assembly and miRNA mediated target mRNA repression. Here, I have observed the
differential compartmentalization of miRNAs and target mRNAs regulated by target
dependent cooperative biogenesis on those cellular compartments.
The other part of work has been concentrated on the study on importance of cellular
membranes on miRNA biogenesis and rER targeting of target mRNA and how this influences
the fate of miRNA. There I have observed reduced compartmentalization of de novo formed
miRNAs on rER or polysomes in defective mitochondria containing cells along with
increased retention of its target mRNAs. I have also observed defective target mRNA
dependent cognate miRNA biogenesis in an amino acid starved and refed hepatic cells.
Increased retention of microsomal target mRNAs in mitochondria-ER detethered cells due to
impaired recycling of miRNP components has been also observed in Mfn2 negative cells.
I have also found a defective intracellular trafficking in growth retarded senescent
mammalian cells having impaired mitochondrial potential and dynamics. Similar to what
happens in senescent cells, Uncoupling Protein 2 mediated depolarization of mitochondrial
membrane potential results in progressive sequestration of miRNAs with polysomes.
Mitochondrial detethering of endoplasmic reticulum, a phenomenon also evident in
mitochondria depolarized cells, found to be responsible for defective compartmentalization of
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translation initiation factor eIF4E to ER attached polysomes. It causes retarded translation
process accompanied by enhanced retention of miRNAs and target mRNAs with rER
attached polysomes that resulted in reduced intracellular trafficking. In subsequent
experiments I have identified a reduced activity of mTORC1 in mitochondria defective cells
to cause reduced phosphorylation of eIF4E-BP1 on microsomes to cause retarded eIF-4E
targeting to ER attached polysome. Cumulatively, these data suggest intricate involvement of
mitochondrial membrane potential and dynamics to determine stability of miRNAs in
mammalian cells by affecting subcellular locations and export of miRNPs. These data
suggest how mitochondrial membrane potential and dynamics, by targeting mTORC1 activity
and compartmentalization, determine subcellular localization of miRNPs. This infers
mitochondrial detethering of rER may directly cause lowering of miRNA turnover by
targeting the initiation phase of translation caused by poor shuttling of cap binding protein
eIF4E from cytoplasmic pool to the rER associated domain.
 
Date 2021
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/2833/1/Susanta_Chatterjee_PhD_Thesis.pdf
CHATTERJEE, SUSANTA (2021) Role of Cellular Membranes in Controlling Micro RNA Mediated Co-ordinated Gene Expression. PhD thesis, University of Calcutta.
 
Relation http://www.eprints.iicb.res.in/2833/