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bioRxiv · 10.1101/2025.07.10.662049

Choreography of human mitochondrial leaderless mRNA translation initiation

Shen, S.·Xu, Y.·Kober, D. L.·Wang, J.
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Abstract

It remains unclear how human mitochondrial ribosomal subunits assemble into an elongation-competent 55S particle on mRNAs devoid of 5 leader sequences. Here, we reconstituted and directly tracked human mitochondrial translation initiation using real-time single-molecule fluorescence spectroscopy. Corroborated with cryo-EM structural analysis, we show that the initiation factor mtIF2 and initiator fMet-tRNAMet are loaded to the 28S subunit to drive mRNAs binding via 5 start codon recognition. This enables sequential loading of the two ribosomal subunits onto the leaderless mRNA to initiate. In parallel, a preassembled 55S monosome can also be loaded with mtIF2 and fMet-tRNAMet to initiate on the mRNA. Both initiation pathways yield active complexes to enter translation elongation, which is gated by mtIF2. The monosome loading pathway can initiate promiscuously with non-formylated Met-tRNAMet, thus its usage may under tight regulation in cells, e.g. by mtIF3. Our work provides a dynamic framework for the distinct human mitochondrial translation initiation.

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Shen, S., Xu, Y., Kober, D. L., Wang, J.. 2025-07-10. Choreography of human mitochondrial leaderless mRNA translation initiation. https://doi.org/10.1101/2025.07.10.662049

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