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http://hdl.handle.net/10603/426107
Title: | Stop codon readthrough in MTCH2 mRNA and its role in mitochondrial physiology |
Researcher: | Lekha, E M |
Guide(s): | Eswarappa, Sandeep M |
Keywords: | Biochemistry and Molecular Biology Biology and Biochemistry Life Sciences |
University: | Indian Institute of Science Bangalore |
Completed Date: | 2020 |
Abstract: | Stop codon readthrough is the process in which translation continues beyond a stop codon till a downstream, in-frame stop codon generating a polypeptide with a C-terminal extension. MTCH2 was selected as a potential readthrough candidate on the basis of evolutionary conservation in the proximal 3and#697;UTR. Here, we demonstrate single and double-stop codon readthrough in MTCH2 mRNA by means of luminescence-based and fluorescence-based assays and by analysing pre-existing ribosome profiling and mass spectrometry data. Our experiments revealed that a 12-nucleotide sequence present in the proximal 3and#697; untranslated region (3and#697;UTR) of MTCH2 can drive both single and double readthrough. Functional characterization of the MTCH2 isoforms revealed that while the canonical protein, MTCH2, and the single readthrough product, MTCH2x, were mitochondrial in nature with long half-life, the double readthrough product, MTCH2xx was found to be short-lived with cytoplasmic localization. HEK293 cells that are MTCH2 readthrough-deficient were generated using CRISPR-Cas9 technique. These cells showed elevated levels of MTCH2 and consistent with this, reduction in mitochondrial membrane potential. Thus, the double stop codon readthrough in MTCH2 regulates its own expression and contributes to the maintenance of normal mitochondrial membrane potential. In a related project, the CRISPR-dCas13a system was used for induction of readthrough. We have targeted the proximal 3and#8242;UTR of MTCH2 mRNA and demonstrated increase in readthrough. Similar strategy was used for the upregulation of readthrough in known readthrough mRNAs AGO1 and VEGFA. CRISPR-dCas13a system was also applied to induce readthrough across the thalassemia-causing premature stop codon in HBB mRNA... newline |
Pagination: | x, 121 |
URI: | http://hdl.handle.net/10603/426107 |
Appears in Departments: | Biochemistry |
Files in This Item:
File | Description | Size | Format | |
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01_title.pdf | Attached File | 145.31 kB | Adobe PDF | View/Open |
02_prelim pages.pdf | 126.26 kB | Adobe PDF | View/Open | |
03_table of content.pdf | 44.93 kB | Adobe PDF | View/Open | |
04_abstract.pdf | 112.19 kB | Adobe PDF | View/Open | |
05_chapter 1.pdf | 654.53 kB | Adobe PDF | View/Open | |
06_chapter 2.pdf | 176.22 kB | Adobe PDF | View/Open | |
07_chapter 3.pdf | 2.07 MB | Adobe PDF | View/Open | |
08_chapter 4.pdf | 1.11 MB | Adobe PDF | View/Open | |
09_chapter 5.pdf | 1.69 MB | Adobe PDF | View/Open | |
10_chapter 6.pdf | 1.14 MB | Adobe PDF | View/Open | |
11_chapter 7.pdf | 943.67 kB | Adobe PDF | View/Open | |
12_annexure.pdf | 224.72 kB | Adobe PDF | View/Open | |
80_recommendation.pdf | 262.79 kB | Adobe PDF | View/Open |
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