The mitochondrial translation machinery highly diverged from its bacterial counterpart. This includes deviation from the universal genetic code, with AGA and AGG codons lacking cognate tRNAs in human mitochondria. The locations of these codons at the end of COX1 and ND6 open reading frames, respectively, suggest they might function as stop codons. However, while the canonical stop codons UAA and UAG are known to be recognized by mtRF1a, the release mechanism at AGA and AGG codons remains a debated issue. Here, we show that upon the loss of another member of the mitochondrial release factor family, mtRF1, mitoribosomes accumulate specifically at AGA and AGG codons. Stalling of mitoribosomes alters COX1 transcript and protein levels, but not ND6 synthesis. In addition, using an in vitro reconstituted mitochondrial translation system, we demonstrate the specific peptide release activity of mtRF1 at the AGA and AGG codons. Together, our results reveal the role of mtRF1 in translation termination at non-canonical stop codons in mitochondria.

Human mitochondria contain a genome (mtDNA) that encodes essential subunits of the oxidative phosphorylation system. Expression of mtDNA entails multi-step maturation of precursor RNA. In other systems, the RNA life cycle involves surveillance mechanisms, however, the details of RNA quality control have not been extensively characterised in human mitochondria. Using a mitochondrial ribosome profiling and mitochondrial poly(A)-tail RNA sequencing (MPAT-Seq) assay, we identify the poly(A)-specific exoribonuclease PDE12 as a major factor for the quality control of mitochondrial non-coding RNAs. The lack of PDE12 results in a spurious polyadenylation of the 3' ends of the mitochondrial (mt-) rRNA and mt-tRNA. While the aberrant adenylation of 16S mt-rRNA did not affect the integrity of the mitoribosome, spurious poly(A) additions to mt-tRNA led to reduced levels of aminoacylated pool of certain mt-tRNAs and mitoribosome stalling at the corresponding codons. Therefore, our data uncover a new, deadenylation-dependent mtRNA maturation pathway in human mitochondria.

Mitochondria harbor specialized ribosomes (mitoribosomes) necessary for the synthesis of key membrane proteins of the oxidative phosphorylation (OXPHOS) machinery located in the mitochondrial inner membrane. To date, no animal model exists to study mitoribosome composition and mitochondrial translation coordination in mammals in vivo. Here, we create MitoRibo-Tag mice as a tool enabling affinity purification and proteomics analyses of mitoribosomes and their interactome in different tissues. We also define the composition of an assembly intermediate formed in the absence of MTERF4, necessary for a late step in mitoribosomal biogenesis. We identify the orphan protein PUSL1, which interacts with a large subunit assembly intermediate, and demonstrate that it is an inner-membrane-associated mitochondrial matrix protein required for efficient mitochondrial translation. This work establishes MitoRibo-Tag mice as a powerful tool to study mitoribosomes in vivo, enabling future studies on the mitoribosome interactome under different physiological states, as well as in disease and aging.

Defects of the translation apparatus in human mitochondria are known to cause disease, yet details of how protein synthesis is regulated in this organelle remain to be unveiled. Here, we characterize a novel human protein, C7orf30 that contributes critically to mitochondrial translation and specifically associates with the large subunit of the mitochondrial ribosome (mt-LSU). Inactivation of C7orf30 in human cells by RNA interference results in respiratory incompetence owing to reduced mitochondrial translation rates without any appreciable effects on the steady-state levels of mitochondrial mRNAs and rRNAs. Ineffective translation in C7orf30-depleted cells or cells overexpressing a dominant-negative mutant of the protein results from aberrant assembly of mt-LSU and consequently reduced formation of the monosome. These findings lead us to propose that C7orf30 is a human assembly and/or stability factor involved in the biogenesis of the large subunit of the mitochondrial ribosome.