Interconnected assembly factors regulate the biogenesis of mitoribosomal large subunit

Abstract

<jats:sec><jats:title>Abstract</jats:title><jats:p>Mitoribosomes synthesize essential proteins encoded in the mitochondrial genome. They consist of the ribosomal RNA and protein components, coordinated assembly of which is critical for function. Divergent mitoribosomes with reduced RNA and increased protein mass were described in <jats:italic>Trypanosoma brucei</jats:italic>, a mammalian parasite causing African Trypanosomiases. Here, we report that the large subunit of <jats:italic>T. brucei</jats:italic> mitoribosome requires at least 15 biogenesis factors, which we characterized in complex with an assembly intermediate using cryo-EM. These assembly factors form a protein network that spans over 180 Å and connects between functional regions, allowing to coordinate their maturation. The conserved proteins mt-RbgA and mt-EngA are stably bound together at the subunit interface and mitochondrial acyl-carrier proteins play a structural role in constraining the L1 stalk. The characterized assembly intermediate forms an extensive network connecting between functionally important regions of the mitoribosome.</jats:p></jats:sec><jats:sec><jats:title>Impact statement</jats:title><jats:p>Biogenesis of <jats:italic>Trypanosoma brucei</jats:italic> mitoribosomal large subunit requires a protein network of assembly factors that connects between its functional regions.</jats:p></jats:sec>

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

“…In bacteria, the MTG1 homolog RbgA binds the LSU in a similar location as MRM2 in our structures, contacting the 23S rRNA at the P- and A-site, and contributing to their maturation 24 . Likewise, Mtg1 in Trypanosoma brucei is bound in complex with the other two GTPases to 12S rRNA in a position similar to the one observed for RbgA 19 , 21 . Based on our structural data, it appears that human MTG1 does not induce pronounced conformational changes of the PTC or participate in the recruitment/dissociation of assembly factors, at the assembly stages captured in this study.…”

“…6b ). Interestingly, the position of human MTG1 in our structures differs significantly from its bacterial and trypanosomal counterparts 19 , 21 , 24 (Supplementary Fig. 6c ).…”

“…Although assembly factors are central to mammalian mitoribosome biogenesis, their molecular mechanisms of action remain largely enigmatic. Indeed, while the assembly pathways of the mt-SSU and mt-LSU in trypanosomes have recently been visualized by cryo-EM 19 – 21 , only one structural study of native human mt-LSU intermediates is available 22 . In these assembly intermediates, over a fifth of the total 16S mt-rRNA (which in mature mitoribosome comprises the PTC and intersubunit bridges) is not yet folded, while the MRP bL36m protein is missing 22 .…”

A distinct assembly pathway of the human 39S late pre-mitoribosome

“…Recently, several kinetoplastid mitoribosomal assembly intermediates were reported, which allow for the comparison of mitoribosome assembly pathways between species 36-38 . Despite the presence of some highly-conserved factors, the kinetoplastid uses numerous species-specific assembly factors which are the main contributors to shape the pre-ribosomal intermediates.…”

“…MALSU1 belongs to the RsfS protein family which, similar to eIF6 in Eukaryotes, binds to uL14 on the large subunit and serves as an anti-association factor preventing premature subunit joining in bacteria 30,35 . Moreover, several large subunit assembly intermediates of kinetoplastid LSU mitoribosome have also been solved by cryo-EM recently 36-38 . Although, we share some of the common AFs with kinetoplastids, the majority of their AFs are species specific and do not exist in human mitochondria.…”