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Translational activators align mitochondrial mRNAs at the small ribosomal subunit for translation initiation

ABSTRACT: Mitochondrial gene expression is essential for oxidative phosphorylation. Mitochondrial-encoded mRNAs are translated by dedicated mitochondrial ribosomes (mitoribosomes), whose regulation remains elusive. In the baker´s yeast Saccharomyces cerevisiae, nuclear-encoded mitochondrial translational activators (TAs) facilitate transcript-specific translation by a yet unknown mechanism. Here, we investigated the function of TAs containing RNA-binding pentatricopeptide repeats (PPRs) using selective mitoribosome profiling and cryo-EM structural analysis. These analyses revealed that TAs exhibit strong selectivity for mitoribosomes initiating on their target transcripts. Moreover, TA-mitoribosome footprints indicated that TAs recruit mitoribosomes proximal to the start codon. Two cryo-EM structures of mRNA-TA complexes bound to post-initiation/pre-elongation-stalled mitoribosomes revealed the general mechanism of TA action. Specifically, the TAs bind to structural elements in the 5' UTR of the client mRNA as well as to the mRNA exit channel to align the mRNA in the small subunit during initiation. Our findings provide a structural basis for understanding how mitochondria achieve transcript-specific translation initiation without relying on general sequence elements to position ribosomes at start codons.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE282943 | GEO | 2025/07/16

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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Translational activators align mitochondrial mRNAs at the small ribosomal subunit for translation initiation

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