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Induction of Transfer RNA Import Into Human Mitochondria : Implications for Correction of Genetic Disorders

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Title Induction of Transfer RNA Import Into Human Mitochondria : Implications for Correction of Genetic Disorders
 
Creator Mahata, Bidesh
 
Subject Molecular & Human Genetics
 
Description A variety of clinical disorders resulted from translation defects due to the mutations in mitochondrial tRNA genes. Till date there is no therapy for these diseases. Here we tried to develop an alternative strategy for such disorders by exploiting RNA import machinery of Leishmania. Our main aim was to pool the cytocolic tRNA to the mitochondrial matrix, of which the mitochondrial cognate is mutated , by introducing RIC into the mitochondrial membrane. Here we have used MERRF (due to A8344G point mutation on mitochondrial tRNALys gene) and Kearns Sayre syndrome( due to deletion of tRNALys along with some other protein coding genes) as model disease and tested our hypothesis on patient derived cybrids homoplasmic for particular mutation.

We have shown here that RIC induces import of human cytoplasmic tRNALys into human isolated mitochondria. The imported tRNA undergoes efficient aminoacylation within the organelle and supports protein synthesis. Mitochondrial translation in MERRF and KSS mitochondria , containing mutant tRNALys genes, is stimulated significantly to near wild type levels and the formation of disease specific aberrant polypeptides suppressed by RIC mediated import of human cytoplasmic tRNALys. When RIC comes in contact with the cells ,it internalized by CAV-1 dependent pathway and targeted to mitochondria. Internalisation process completed within 24 hour of RIC treatment to the cells. We found RIC treated cells import specific tRNAs which contain the import signal including tRNALys from cytosol. Such RIC induced import is able restore the lost mitochondrial membrane potential,depleted mitochondrial translation and lost Complex IV activity in MERRF cells .RIC treated MERRF cells grows normally in galactose containing media indicating restoration of lost respiratory chain. Moreover oxygen consumption rateof RIC treated MERRF cells reaches near wild type level. In KSS cell the treatment of RIC has no effect on mitochondrial function as along with tRNALys , the COII, COIII, A6 and A8 gene are also deleted. But expession of all the existing protein coding genes increases due to import of cytosolic tRNAs. We did not found any cytotoxic/apoptotic effect of RIC in cultured cells and grows normally in presence of RIC. These results suggest a novel way to introduce a heterologous tRNA import machinery for correction of disorders due to mitochondrial tRNA mutations. Here we have shown that in case of tRNALys point mutation it is effective in vitro / in live cells.. We demonstrated here that besides tRNALys some other tRNAs containing signal motif were imported, among them some are disease causing when mutated. So this approach could also be applied to other disease causing mitochondrial tRNA mutations.
 
Date 2007
 
Type Thesis
NonPeerReviewed
 
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Identifier http://www.eprints.iicb.res.in/1597/1/COVER_PAGE.doc
http://www.eprints.iicb.res.in/1597/2/ACKNOWLEDGEMENT.doc
http://www.eprints.iicb.res.in/1597/3/ABBREVIATIONS.doc
http://www.eprints.iicb.res.in/1597/4/Abstract507(within350_words)Bidesh.doc
http://www.eprints.iicb.res.in/1597/5/synopsis.doc
http://www.eprints.iicb.res.in/1597/6/CONTENTS.doc
http://www.eprints.iicb.res.in/1597/7/Starting_pages.doc
http://www.eprints.iicb.res.in/1597/8/BidThesIntro.doc
http://www.eprints.iicb.res.in/1597/9/BidThesMaterialMethods.doc
http://www.eprints.iicb.res.in/1597/10/BidThesRESULT.doc
http://www.eprints.iicb.res.in/1597/11/thesis_bidesh_DISCUSSion.doc
http://www.eprints.iicb.res.in/1597/12/REFERENCES.doc
http://www.eprints.iicb.res.in/1597/13/List_of__Publications.doc
Mahata, Bidesh (2007) Induction of Transfer RNA Import Into Human Mitochondria : Implications for Correction of Genetic Disorders. PhD thesis, Calcutta University.
 
Relation http://www.eprints.iicb.res.in/1597/