Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 295, Issue 50, Pages 17009-17026Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA120.014253
Keywords
mitochondria; MTCH2; stop codon; translational read-through; translation control; mRNA; protein degradation; mitochondrial membrane potential; ribosome; stop; codon
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Funding
- Department of Biotechnology/Wellcome Trust India Alliance Fellowship Grant [IA/I/15/1/501833]
- Department of Biotechnology-Indian Institute of Science Partnership Program for Advanced Research in Biological Sciences Grant [BT/PR27952-INF/22/212/2018]
- University Grants Commission, India
- Council of Scientific & Industrial Research-University Grants Commission, India
- Kishore Vaigyanik Protsahan Yojana fellowship from the Department of Science and Technology, India
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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 3 ' 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 3 ' UTR of MTCH2 is the necessary signal for SCR. Functional characterization showed that MTCH2 and MTCH2x were localized to mitochondria with a long t(1/2) (>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 t(1/2) (<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 normal mitochondrial membrane potential.
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