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Distinct roles for human mtIF2 and mtIF3 in non-canonical mitochondrial translation.

ABSTRACT: The synthesis of mitochondrial OXPHOS complexes is central to cellular metabolism, yet many molecular details of mitochondrial translation remain elusive. It is commonly held view that translation initiation in human mitochondria proceeded in a manner similar to bacterial systems, with the mitoribosomal small subunit bound to the initiation factors, mtIF2 and mtIF3, along with initiator tRNA and an mRNA. However, unlike in bacteria, most human mitochondrial mRNAs lack 5′ leader sequences that can mediate small subunit binding, raising the question of how leaderless mRNAs are recognized by mitoribosomes. By using novel in vitro mitochondrial translation initiation assays, alongside biochemical and genetic characterization of cellular knockouts of mitochondrial translation factors, we describe unique features of translation initiation in human mitochondria. We show that in vitro, leaderless mRNA transcripts can be loaded directly onto assembled 55S mitoribosomes, but not onto the mitoribosomal small subunit (28S). In addition, we demonstrate that while mtIF2 is indispensable for mitochondrial translation, mtIF3 activity is not required for translation of leaderless mitochondrial transcripts but is essential for translation of ATP6 in the case of the bicistronic ATP8/ATP6 transcript. Our results confirm important evolutionary divergences of the mitochondrial translation system, and further our understanding of a process central to eukaryotic metabolism.

ORGANISM(S): Homo sapiens

PROVIDER: GSE156937 | GEO | 2022/12/01

REPOSITORIES: GEO

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Distinct roles for human mtIF2 and mtIF3 in non-canonical mitochondrial translation

Project description:The production of mitochondrial OXPHOS complexes is central to cellular metabolism, although the molecular details of mitochondrial translation remain enigmatic. It is widely held that translation initiation in human mitochondria proceeds similarly to bacterial systems, with mRNA binding the mitoribosomal small subunit in the presence of initiation factors, mtIF2and mtIF3, and initiator tRNA. However, unlike in bacteria, most human mitochondrial mRNAs do not possess 5′ leader sequences that mediate binding to the small subunit. Thus, how leaderless mRNAs are recognized by the mitoribosome is not known. By developing a single-molecule, fluorescence-based in vitro translation initiation assay, alongside the biochemical and genetic characterization of cellular knockouts of mitochondrial translation factors, we describe a mechanism for non-canonical translation initiation in human mitochondria. We show leaderless mt-mRNAs can be loaded onto 55S monosomes and translated independently of mtIF3 activity. However, in the case of the bicistronic ATP8/ATP6 transcript, translation of the downstream open reading frame (ORF) is dependent upon mtIF3 and is uncoupled from the upstream leaderless ORF, highlighting distinct role for the human initiation factor. Furthermore, we found mtIF2 to be essential for mitochondrial protein synthesis, but not monosome formation, while mitoribosome recycling was important for mitoribosome homeostasis. These data define an important evolutionary diversion of mitochondrial translation system, and further our fundamental understanding of a process central to eukaryotic metabolism.

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