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Monitoring the complexity and dynamics of mitochondrial translation

ABSTRACT: Eukaryotes house the additional protein synthesis system in mitochondria. Given the mitochondrial translation dictates OXPHOS complex abundance and then ATP level in cells, the exhaustive quantitative, and high-resolution delineation of mitoribosome traversal has been awaited. Here, we developed a high-resolution mitochondrial ribosome profiling and unveiled tight regulation of in organello translation. Our approach assessed the stoichiometry and kinetics of mitochondrial translation flux, such as the absolute mitoribosome numbers on a transcript, elongation rate, initiation rate, and lifetime translation round on a transcript. We also surveyed the impacts of all the 34th position modifications on mt-tRNAs, including f5C34, m5C34, τm5U34, τm5s2U34, s2U34, and Q34, as well as t6A37 by the responsible enzyme deletions. Moreover, retapamulin-assisted profiling and disome profiling unveiled cryptic translation initiation sites at sub-cognate codons and the programmed mitoribosome collision sites across the mitochondrial transcriptome, respectively. Our work provides a useful resource to delineate protein synthesis within the bioenergetic powerhouse.

ORGANISM(S): Mus musculus Mus Homo sapiens

PROVIDER: GSE237154 | GEO | 2025/10/22

REPOSITORIES: GEO

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Project description:Mitochondrial ribosomes (mitoribosomes) have undergone substantial structural remodelling throughout evolution leading to distinct structural properties compared to their prokaryotic ancestors. Compared to their prokaryotic counterparts, mitoribosomes show a substantial loss of ribosomal RNA (rRNA), whilst acquiring unique mitochondrial proteins located on the periphery of the mitoribosome. This extended protein content suggests mitochondrial-specific functions of these proteins involved in mitochondrial mRNA selective-translation activation, ribosome assembly, translation regulation rather than exclusively structural scaffolding. We investigated the functional properties of the 14 unique (mitochondria-specific or supernumerary) mammalian mitoribosomal proteins (snMRP) in the small mitochondrial subunit (mtSSU) by CRISPR-mediated knockout and describe their specific effect on mitoribosome integrity and function. Unexpectedly, we show that each snMRP knockout leads to a unique pattern of mitoribosome instability with variable effects on mitochondrial protein synthesis as essential structural components and all – with the exception of mS37 (MRPS37, CHCHD1) – to almost complete loss of protein synthesis. Concomitantly, through steady-state proteomic analysis we confirm a modular assembly of the mtSSU. Surprisingly, mS37 was found to have impaired stability in all our tested mtSSU and large mitochondrial subunit (mtLSU) snMRPs knockouts as well as in patient-derived cell lines, suggesting an extended role of the protein in mtSSU pre-translation initiation steps. As mitochondrial intermembrane (IMS) targeted protein – a eukaryotic-confined localisation mechanism – with additional unique MIA40 (CHCHD4)-oxidisable motifs involved in the disulfide relay system, mS37 could have a specific role in mitochondrial translation regulation. Indeed, we find oxidation of the cysteines in the CX9C motif of mS37 to be essential for its stability, confirming mS37 regulating mitochondrial translation regulation.

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Monitoring the complexity and dynamics of mitochondrial translation

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