Pervasive non-stop mRNAs generate the ground state of mitochondrial gene expression noise

A stop codon within the mRNA facilitates coordinated termination of protein synthesis, releasing the nascent polypeptide chain from the ribosome. This essential step in gene expression is impeded with transcripts lacking a stop codon, generating non-stop ribosome complexes. The frequency by which these mRNA events arise remains a key question in the regulation of gene expression. Here, we used deep sequencing to investigate the quantitative and qualitative nature by which non-stop mRNAs are generated from the human mitochondrial genome. We reveal an extensive level of diverse non-stop mRNAs on mitochondrial ribosomes that are resistant to translation termination by canonical release factors. Instead, rescue of stalled non-stop ribosome complexes requires the ribosome quality control factor C12orf65 (mtRF-R). Furthermore, our analysis indicates that mRNAs with the AGA and AGG codons, which lack recognition by a cognate tRNA, should be reappraised as non-stop mRNAs. Lastly, we show that the failure to resolve these gene expression aberrations imparts a negative regulatory effect on protein synthesis, revealing a molecular basis for the deleterious effect of specific pathogenic variants expressed from the mitochondrial genome. Collectively, our findings reveal the underlying noise in mitochondrial gene expression and the importance of response quality control mechanisms for cell fitness.