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  1. Evaluation Summary:

    Ribosomes are among the most complex molecular machines a cell makes. The work by Chandrasekaran et al. contributes to our understanding of the molecular details of mitochondrial ribosome assembly, and how disruptions to this pathway may cause human disease. Using cryo-EM, the authors identified a subpopulation of immature human mitochondrial large ribosomal subunits that interact with assembly factors NSUN4, MTERF4 and GTPBP7. Based on this structure, they introduce mutations in C. elegans orthologs of these assembly factors that are expected to disrupt binding to the large subunit, and they show that these mutants cause sterility and disrupt mitochondrial proteostasis in the mutant animals. The work does not yet establish a direct link between the putative structural defects of the mutants and the observed developmental defects in C. elegans. Additional studies are needed to test the interesting possibility that this structure represents an on-pathway intermediate of mitoribosome biogenesis and/or ribosome recycling.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their names with the authors.)

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  2. Reviewer #1 (Public Review):

    The authors present a cryo-EM structure of an assembly intermediate of the human mitochondrial ribosome (mitoribosome) purified in the presence of GMPPCP to inhibit GTPBP7, one of the GTPases involved in the assembly of the large ribosomal subunit, thereby stabilizing the structure. The trapped intermediate shows clear density for assembly factors NSUN4 and MTERF4, an RNA methyltransferase and transcription termination factor, respectively, both of which are important for the correct folding of the 16S ribosomal RNA. The spatial resolution achieved by the authors is sufficient to pinpoint some of the molecular interactions, at the amino acid side chain level, that are functionally relevant. These details, and correlating point mutations at different interaction sites with developmental defects in C. elegans, provide strong support for the authors' proposed role of NSUN4 and MTERF4.

    Nevertheless, the use of the non-hydrolysable GTP analog, GMPPCP, as well as purification of intermediates known to be transient introduce some uncertainty in the relevance of the observed structures. While showing that point mutations at interaction sites have a phenotype in C. elegans, structural details of the interactions visualized in the trapped intermediate may still be different under native conditions.

    Overall, this work adds important structural detail to our understanding of how the peptidyl transferase center folds correctly, an essential step in the assembly pathway of mitoribosomes. Insights gained by the authors may also be relevant for the biogenesis pathway of cytoplasmic ribosomes, and they will help understand disease mechanism associated with defective mitoribosome biogenesis.

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  3. Reviewer #2 (Public Review):

    This paper is one of several recently released studies describing presumed late intermediates of mitochondrial LSU biogenesis. The authors of this study are experts in ribosome structure determination and RNA processing in C. elegans. In the course of another study to interrogate mitochondrial translation, the authors purified mitochondria from PDE12-/- HEK293T cells that show defects in translation and isolated mitochondrial ribosomal subunits by sucrose gradient preparation in the presence of the non-hydrolysable GTP analog, GMPPCP. They identified a sub-population of mitoribosome large subunits with H68-71 of the 16S rRNA in a partially unfolded state and the proteins NSUN4, MTERF4 and GTPBP7 bound that is incompatible with translation. This structure is potentially interesting because it presents a plausible new intermediate of mitoribosome biogenesis and a possible mechanism for ribosome quality control during recycling.

    The cryo-EM map presented is consistent with an open unfolded conformation of H68-71 and the binding sites of NSUN4, MTERF4 and GTPBP7 as the authors propose. The binding sites of NSUN4 and MTERF4 are also consistent those presented in 2 other concurrent studies, providing further support for this interaction interface. Unlike the concurrent studies by Hillen et al., and Cipullo et al., the LSU is close to fully mature in this structure, making interpretation of the precise role(s) of NSUN4 and MTERF4 in mitoribosome maturation complex. The binding location of GTPBP7 in this model is also different from that observed by Hillen et al., and Cipullo et al., but is consistent with the binding site of the bacterial ortholog RbgA, which can also function in mitoribosome biogenesis, providing evolutionary support for the observed binding site.

    The authors found that mutations in the C. elegans orthologs of MTERF4 and GTPBP7 predicted from their model to interrupt the interaction with 16S rRNA cause sterility, mitochondrial proteotoxic stress and, for MTERF4, developmental delays. However, the authors did not confirm that these mutants interrupt the interaction with the LSU and that the mutant proteins were expressed at or above the level of their wt counterparts. This makes it difficult to determine whether the observed physiological effects were due to loss of this interaction or down-regulation of the proteins.

    The authors' suggestion that this complex might form during mitoribosome recycling is intriguing, especially since this complex was isolated from PDE12-/- cells which show translational defects. As the authors acknowledge, this role for NSUN4, MTERF4 and GTPBP7 is at this point speculative.

    In summary, this study presents a plausible intermediate of mitoribosome biogenesis, highlighting potential roles for NSUN4, MTERF4 and GTPBP7 in late mitoribosome biogenesis and speculatively mitoribosome quality control. Further work is required to determine whether the complex presented represents an on-pathway intermediate of mitoribosome assembly in vivo.

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  4. Reviewer #3 (Public Review):

    The overall goal of this study was to capture structures of the assembly intermediates of the human large mitoribosomal subunit that involve GTPases. Authors used a non-hydrolysable GTP analog to arrest the bound form of assembly factor(s) that would otherwise be released in the absence of a GTP analog. While authors succeeded in capturing high-resolution structural intermediates, they failed to some extent in establishing a direct correlation with the point mutations and the biochemical studies carried out in a model organism C. elegans. At times the two studies seem to be disconnected.

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