Structural basis for late maturation steps of the human mitoribosomal large subunit

Abstract

Mitochondrial ribosomes (mitoribosomes) synthezise a critical set of proteins essential for oxidative phosphorylation. Therefore, their function is vital to cellular energy supply and mitoribosomal defects give rise to a large and diverse group of human diseases 1. The architecture of mitoribosomes is strikingly different from that of their bacterial and eukaryotic cytosolic counterparts and display high divergence between species 2-6. Mitoribosome biogenesis follows distinct molecular pathways that remain poorly understood. Here, we determined the cryo-EM structures of mitoribosomes isolated from human cell lines with either depleted or overexpressed mitoribosome assembly factor GTPBP5. This allowed us to capture consecutive steps during mitoribosomal large subunit (mt-LSU) biogenesis that involve normally short-lived assembly intermediates. Our structures provide important insights into the last steps of 16S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, which require the coordinated action of nine assembly factors. We show that mammalian-specific MTERF4 contributes to the folding of 16S rRNA, allowing 16S rRNA methylation by MRM2, while GTPBP5 and NSUN4 promote fine-tuning rRNA rearrangments leading to PTC formation. Moreover, our data reveal an unexpected role for the elongation factor mtEF-Tu in mt-LSU assembly, in which mt-EF-Tu interacts with GTPBP5 in a manner similar to its interaction with tRNA during translational elongation. Together, our approaches provide detailed understanding of the last stages of mt-LSU biogenesis that are unique to mammalian mitochondria.