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Stop codon read-through of mammalian MTCH2 leading to an unstable isoform regulates mitochondrial membrane potential

Manjunath, LE and Singh, A and Sahoo, S and Mishra, A and Padmarajan, J and Basavaraju, CG and Eswarappa, SM (2020) Stop codon read-through of mammalian MTCH2 leading to an unstable isoform regulates mitochondrial membrane potential. In: Journal of Biological Chemistry, 295 (50). pp. 17009-17026.

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Official URL: https://doi.org/10.1074/jbc.RA120.014253


Stop codon read-through (SCR) is a process of continuation of translation beyond a stop codon. This phenomenon, which occurs only in certain mRNAs under specific conditions, leads to a longer isoform with properties different from that of the canonical isoform. MTCH2, which encodes a mitochondrial protein that regulates mitochondrial metabolism, was selected as a potential read-through candidate based on evolutionary conservation observed in the proximal region of its 39 UTR. Here, we demonstrate translational read-through across two evolutionarily conserved, in-frame stop codons of MTCH2 using luminescence- and fluorescence-based assays, and by analyzing ribosome-profiling and mass spectrometry (MS) data. This phenomenon generates two isoforms, MTCH2x and MTCH2xx (single- and double-SCR products, respectively), in addition to the canonical isoform MTCH2, from the same mRNA. Our experiments revealed that a cis-acting 12-nucleotide sequence in the proximal 39 UTR of MTCH2 is the necessary signal for SCR. Functional characterization showed that MTCH2 and MTCH2x were localized to mitochondria with a long t1/2(>36 h). However, MTCH2xx was found predominantly in the cytoplasm. This mislocalization and its unique C terminus led to increased degradation, as shown by greatly reduced t1/2(<1 h). MTCH2 read-through-deficient cells, generated using CRISPR-Cas9, showed increased MTCH2 expression and, consistent with this, decreased mitochondrial membrane potential. Thus, double-SCR of MTCH2 regulates its own expression levels contributing toward the maintenance of normalmitochondrialmembrane potential. © 2020 American Society for Biochemistry and Molecular Biology Inc.. All rights reserved.

Item Type: Journal Article
Publication: Journal of Biological Chemistry
Publisher: American Society for Biochemistry and Molecular Biology Inc.
Additional Information: The copyright for this article belongs to The American Society for Biochemistry and Molecular Biology Inc.
Keywords: Cytology; Magnesium printing plates; Mammals; Mass spectrometry, Cis actings; Evolutionary conservations; Expression levels; Functional characterization; Mislocalization; Mitochondrial membrane potential; Mitochondrial metabolism; Mitochondrial protein, Mitochondria, 3' untranslated region; amino acid sequence; animal cell; animal tissue; Article; carboxy terminal sequence; cell culture; cell isolation; cell proliferation assay; controlled study; CRISPR-CAS9 system; cytoplasm; fluorescence; gene; gene control; gene deletion; gene expression; gene location; genetic conservation; human; human cell; luminescence; mammal; mass spectrometry; mitochondrial membrane potential; mouse; MTCH2 gene; nonhuman; nucleotide sequence; priority journal; real time polymerase chain reaction; ribosome; RNA isolation; RNA translation; stop codon; Western blotting; animal; aorta; bovine; CRISPR Cas system; cytology; genetics; HEK293 cell line; metabolism; protein synthesis; stop codon; vascular endothelium, carrier protein; isoprotein; messenger RNA; MTCH2 protein, human, 3' Untranslated Regions; Animals; Aorta; Base Sequence; Cattle; Codon, Terminator; CRISPR-Cas Systems; Endothelium, Vascular; HEK293 Cells; Humans; Membrane Potential, Mitochondrial; Mitochondrial Membrane Transport Proteins; Protein Biosynthesis; Protein Isoforms; Ribosomes; RNA, Messenger
Department/Centre: Division of Biological Sciences > Biochemistry
Date Deposited: 09 Jan 2023 09:24
Last Modified: 09 Jan 2023 09:24
URI: https://eprints.iisc.ac.in/id/eprint/78946

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