Human mtRF1 terminates COX1 translation and its ablation induces mitochondrial ribosome-associated quality control

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Abstract

Translation termination requires release factors that read a STOP codon in the decoding center and subsequently facilitate the hydrolysis of the nascent peptide chain from the peptidyl tRNA within the ribosome. In human mitochondria eleven open reading frames terminate in the standard UAA or UAG STOP codon, which can be recognized by mtRF1a, the proposed major mitochondrial release factor. However, two transcripts encoding for COX1 and ND6 terminate in the non-conventional AGA or AGG codon, respectively. How translation termination is achieved in these two cases is not known. We address this long-standing open question by showing that the non-canonical release factor mtRF1 is a specialized release factor that triggers COX1 translation termination, while mtRF1a terminates the majority of other mitochondrial translation events including the non-canonical ND6. Loss of mtRF1 leads to isolated COX deficiency and activates the mitochondrial ribosome-associated quality control accompanied by the degradation of COX1 mRNA to prevent an overload of the ribosome rescue system. Taken together, these results establish the role of mtRF1 in mitochondrial translation, which had been a mystery for decades, and lead to a comprehensive picture of translation termination in human mitochondria.

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  1. Review coordinated via ASAPbio’s crowd preprint review

    This review reflects comments and contributions by Ruchika Bajaj, Pablo Ranea-Robles and Michael Robichaux. Review synthesized by Michael Robichaux.


    The manuscript reports findings from new knockout human cell lines for the mitochondrial release factors mtRF1 and mtRF1a. The work contributes new insight into mitochondrial protein translation and mechanisms related to mitochondrial disease. A specific role is demonstrated for the release factor mtRF1 in the translation of COX1, a mitochondrial respiratory protein. The manuscript also identified a compensatory role for the mitochondrial ribosome-associated quality control (mtRQC) pathway when mitochondrial translation termination is impaired.

    Overall the experiments and results presented in the manuscript are supportive of the conclusions described in the text. These findings are impactful toward understanding mitochondrial translation termination.

    Major comments

    • In the results section related to Figure 1d, an increase in reactive oxygen species (ROS) is measured using the mitoSOX probe. Considering that mitoSOX measures superoxide accumulation in the mitochondria, please consider specifying in the text that the ROS measured is of mitochondrial origin. In addition, since mitoSOX labeling may be affected by changes in mitochondrial membrane potential or mitochondrial shape and size, please consider adding an experimental condition using a membrane-potential-responsive, redox-insensitive probe. Finally, please clarify the results presented in Figure 1d with more technical detail. What do the n-values signify? Technically, how is ROS production measured?
    • For Figure 2b-d, in gel activity for complex I and IV are measured; please provide further technical details for these experiments. Please describe what kind of activity is being measured and how it is measured. Also consider adding a density graph of these gel data for clarification of the results.
    • Referencing Figure 2b-c, specifically, it is stated in the Results section that “mtRF1 loss does not affect complex I..”; however, the figure shows an increase in activity of ~20% for the mtRF1-/- condition. Please consider rephrasing or clarifying this point.
    • For Figure 3, there is a possible discrepancy in these results that may need to be addressed. For example, the difference of the relative intensity of ND6 between the WT vs. mtRF1a-/- conditions shown in Fig 3a is significantly less than what is quantified for the same comparison in the bar graph in Fig 3e. It is possible these analyses were performed differently; if so, please report this.

    Minor comments

    • It is stated in the first Results section: “In the absence of mtRF1a, cells tend to produce more reactive oxygen species (ROS)...”, which is vague, please rewrite more technically since it is describing the quantitative data in Fig 1D. From this same section, the final statement: “Thus, both release factors are critical for mitochondrial function and cellular growth” is perhaps too conclusive based only on the results from Figure 1.
    • Related to Fig 1c: consider converting the graph to a log scale, which may help illustrate the difference in growth rates between conditions. In addition to measuring cellular growth, please also consider measuring/counting mitochondria and examining cell morphology changes, which may be easy, additive experiments to include here.
    • In the Results section related to Fig. 2a-d, the respiratory chain complexes are presented with no context. Consider mentioning these complexes in the Introduction or contextualizing them better in this section.
    • In this same Results section please add appropriate citations for “Figure 2g” when referencing results related to that figure panel.
    • A portion of the results text related to Figure 4a-b states: “With the exception of MTND6 (mRNA encoding for ND6), all of the mt-mRNAs arise from the polycistronic transcript synthesized from the heavy strand. If the loss of mitochondrial RFs would affect mitochondrial transcription, one would expect an overall decrease in all mitochondrial transcripts. However, as we observe a selective decrease in specific transcripts in the individual knockouts, we conclude that it is more likely an issue of RNA stability rather than synthesis.” This may be more appropriate to include in the Discussion section.
    • In the Results section related to Figure 5, please again consider properly citing the figures when describing the results presented in those figures and panels.
    • While Figure 6 is an informative model figure, please consider explaining the model with respect to results in the manuscript.

    Comments on reporting

    • Please consider adding more detail in the Methods section about the statistical analyses performed in this study. In addition, other statistical tests may be needed for some group comparisons (e.g., two-way ANOVA for the data in Fig. 5d).
    • For all the western blot data presented in the manuscript, please consider adding the full blot scans to the supplemental material.
    • Referencing Supplementary Table S1, please consider adding validation references for the antibodies used in this study. This is of great benefit to other researchers.

    Suggestions for future studies

    Future studies may test the effect of the combined ablation of the mtRF1 and mtRF1a release factors.