Visualizing formation of the active site in the mitochondrial ribosome.

Viswanathan Chandrasekaran,Nicholas O Burton,Nirupa Desai,Hanting Yang,Eric A Miska,V Ramakrishnan,Jon Price

doi:10.7554/elife.68806

Viswanathan Chandrasekaran, Nicholas O Burton + Show 5 more

Open Access

https://doi.org/10.7554/elife.68806

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Abstract

Ribosome assembly is an essential and conserved process that is regulated at each step by specific factors. Using cryo-electron microscopy (cryo-EM), we visualize the formation of the conserved peptidyl transferase center (PTC) of the human mitochondrial ribosome. The conserved GTPase GTPBP7 regulates the correct folding of 16S ribosomal RNA (rRNA) helices and ensures 2'-O-methylation of the PTC base U3039. GTPBP7 binds the RNA methyltransferase NSUN4 and MTERF4, which sequester H68-71 of the 16S rRNA and allow biogenesis factors to access the maturing PTC. Mutations that disrupt binding of their Caenorhabditis elegans orthologs to the large subunit potently activate mitochondrial stress and cause viability, development, and sterility defects. Next-generation RNA sequencing reveals widespread gene expression changes in these mutant animals that are indicative of mitochondrial stress response activation. We also answer the long-standing question of why NSUN4, but not its enzymatic activity, is indispensable for mitochondrial protein synthesis.

Highlights

NSUN4MTERF4 and GTPBP7 during mitoribosome biogenesis, in vivo mutagenesis designed to disrupt binding of their Caenorhabditis elegans orthologs to the large subunit potently activates mitochondrial stress responses and results in severely reduced viability, developmental delays and sterility
Mutations that disrupt mitoribosome binding to MTER-4 and MTG-1, led to a substantial increase in expression of the mitochondrial heat-shock protein HSP-6, consistent with activation of the mitochondrial unfolded protein response (Fig. 3a)
Our results suggest that the effects of disrupting GTPBP7/MTG-1 and mitochondrial transcription termination factor 4 (MTERF4)/MTER-4 interactions with the mitoribosome can be overcome in somatic tissues, potentially by activating the mitochondrial unfolded protein response, as mutant animals can develop to adulthood

Summary

Ribosome assembly is an essential and complex process that is regulated at each step by specific biogenesis factors. Mutations that disrupt mitoribosome binding to MTER-4 and MTG-1 (the C. elegans orthologs of MTERF4 and GTPBP7 respectively), led to a substantial increase in expression of the mitochondrial heat-shock protein HSP-6, consistent with activation of the mitochondrial unfolded protein response (Fig. 3a) These findings are similar to the effects observed when mitochondrial ribosomal proteins are knocked down by RNAi18. Our observations of severe mitochondrial defects and activation of the mitochondrial unfolded protein response in mter-4 mutants, but not nsun-4 catalytic dead mutants, suggests that the major function of these proteins is as a checkpoint in mitochondrial ribosome assembly and not in RNA methylation Consistent with this idea, MTERF4 does not participate during. We have shown, using structure-directed mutagenesis in vivo, that while the methyltransferase activity of NSUN4 on the small subunit can be dispensed with without resulting in a strong phenotype, the protein has a more important non-enzymatic function as a biogenesis factor in the final stages of the formation of the PTC and the tRNA binding sites in the large subunit

A Scheme for the assembly of the peptidyl transferase centre

Findings

Methods