Determining the off-target activity of antibiotics and novel translation initiation sites in mitochondria

  1. James Marks
  2. Emma Young
  3. Markus Hafner

  • Curated by eLife

    eLife logo

    eLife Assessment

    This valuable study presents findings on how prokaryotic antibiotics affect translation in mitochondrial ribosomes. Using mitoribosome profiling, the authors provide solid evidence that most tested antibiotics act similarly on bacterial and mitochondrial translation. Additionally, this work shows that alternative translation initiation events might exist in two specific mt-mRNAs (MT-ND1 and MT-ND5). However, additional biochemical and structural experiments are needed to support these findings.

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Abstract

Mitochondria translate the 13 mitochondrially encoded proteins using a dedicated translation system that closely resembles the one found in prokaryotes. Consequently, many bacterial protein synthesis inhibitors, used as antibiotics, exhibit mitochondrial toxicity as off-target effects. However, whether these antibiotics act through the same mechanisms in mitochondria as in bacteria remains unclear. To address this, we characterized the impact of a panel of bacterial translation and elongation inhibitors on mitochondrial translation through mitoribosome profiling. We found that the mechanism of action for every antibiotic, except telithromycin, is the same in both bacteria and mitochondria. Additionally, mitoribosome profiling data showed that MT-ND1 and MT-ND5 have incorrectly annotated translation initiation sites and suggested several novel mitochondrial translation events. Careful study of the mechanisms of mitochondrial translation inhibition can guide development of antibiotics with increased target specificity and reduced mitochondrial toxicity.

Article activity feed

  1. eLife

    eLife Assessment

    This valuable study presents findings on how prokaryotic antibiotics affect translation in mitochondrial ribosomes. Using mitoribosome profiling, the authors provide solid evidence that most tested antibiotics act similarly on bacterial and mitochondrial translation. Additionally, this work shows that alternative translation initiation events might exist in two specific mt-mRNAs (MT-ND1 and MT-ND5). However, additional biochemical and structural experiments are needed to support these findings.

  2. eLife

    Reviewer #1 (Public review):

    Summary:

    This study aimed to determine whether bacterial translation inhibitors affect mitochondria through the same mechanisms. Using mitoribosome profiling, the authors found that most antibiotics, except telithromycin, act similarly in both systems. These insights could help in the development of antibiotics with reduced mitochondrial toxicity.

    They also identified potential novel mitochondrial translation events, proposing new initiation sites for MT-ND1 and MT-ND5. These insights not only challenge existing annotations but also open new avenues for research on mitochondrial function.

    Strengths:

    Ribosome profiling is a state-of-the-art method for monitoring the translatome at very high resolution. Using mitoribosome profiling, the authors convincingly demonstrate that most of the analyzed antibiotics act in the same way on both bacterial and mitochondrial ribosomes, except for telithromycin. Additionally, the authors report possible alternative translation events, raising new questions about the mechanisms behind mitochondrial initiation and start codon recognition in mammals.

    Weaknesses:

    All the weaknesses I previously highlighted were adequately addressed.

  3. eLife

    Reviewer #3 (Public review):

    Summary:

    Recently, the off-target activity of antibiotics on human mitoribosome has been paid more attention in the mitochondrial field. Hafner et al applied mitoribosome profilling to study the effect of antibiotics on protein translation in mitochondria as there are similarities between bacterial ribosome and mitoribosome. The authors conclude that some antibiotics act on mitochondrial translation initiation by the same mechanism as in bacteria. On the other hand, the authors showed that chloramphenicol, linezolid and telithromycin trap mitochondrial translation in a context-dependent manner. More interesting, during deep analysis of 5' end of ORF, the authors reported the alternative start codon for ND1 and ND5 proteins instead of previously known one. This is a novel finding in the field and it also provide another application of the technique to further study on mitochondrial translation.

    Strengths:

    This is the first study which applied mitoribosome profiling method to analyze mutiple antibiotics treatment cells. The mitoribosome profiling method had been optimized carefully and has been suggested to be a novel method to study translation events in mitochondria. The manuscript is constructive and well-written.

    Weaknesses:

    This is a novel and interesting study, however, most of conclusion comes from mitoribosome profiling analysis, as the result, the manuscript lacks the cellular biochemical data to provide more evidence and support the findings.

    Comments on revisions:

    The authors addressed most of my concerns and comments, although there is still no biochemical assay which should be performed to support mitoribsome profiling data.

    The author also carefully investigated the structure of complex I, however, I am surprised that the author chose to analyse a low resolution structure (3.7 A). Recently, there are more high resolution structures of mammalian complex I published (7R41, 7V2C, 7QSM, 9I4I). Furthermore, the authors should not only respond to the reviewers but also (somehow) discuss these points in the manuscript.

  4. eLife

    Author response:

    The following is the authors’ response to the original reviews.

    Public Reviews:

    Reviewer #1 (Public review):

    Summary:

    This study aimed to determine whether bacterial translation inhibitors affect mitochondria through the same mechanisms. Using mitoribosome profiling, the authors found that most antibiotics, except telithromycin, act similarly in both systems. These insights could help in the development of antibiotics with reduced mitochondrial toxicity.

    They also identified potential novel mitochondrial translation events, proposing new initiation sites for MT-ND1 and MT-ND5. These insights not only challenge existing annotations but also open new avenues for research on mitochondrial function.

    Strengths:

    Ribosome profiling is a state-of-the-art method for monitoring the translatome at very high resolution. Using mitoribosome profiling, the authors convincingly demonstrate that most of the analyzed antibiotics act in the same way on both bacterial and mitochondrial ribosomes, except for telithromycin. Additionally, the authors report possible alternative translation events, raising new questions about the mechanisms behind mitochondrial initiation and start codon recognition in mammals.

    Weaknesses:

    The main weaknesses of this study are:

    While the authors highlight an interesting difference in the inhibitory mechanism of telithromycin on bacterial and mitochondrial ribosomes, mechanistic explanations or hypotheses are lacking.

    We acknowledge that we were not able to present a clear explanation for potential mechanistic differences of telithromycin inhibition between mitochondrial and bacterial ribosomes. In future work, structural analyses in collaboration with experts will provide these insights.

    The assignment of alternative start codons in MT-ND1 and MT-ND5 is very interesting but does not seem to fully align with structural data.

    We appreciate the reviewer’s comment and consulted a cryo-EM expert to review our findings in the context of the available structural data. We downloaded the density map and reviewed the N-termini of MT-ND1 and MT-ND5. We only observed the density of the N-terminus of MT-ND1 at low confidence. At an RMSD of 2, we could not observe density for the side chains of Met and Pro, and there are gaps in the density for what is modeled as the main chain. The assignment of these residues may have been overlooked due to the expectation that they should be present in the peptide.

    For MT-ND5, we did observe some density that could be part of the main chain; however, it did not fill out until we reduced the stringency, and we did not observe density mapping to side chain residues. To summarize, we do not confidently see density for either the side chain or the main chain for either peptide.

    The newly proposed translation events in the ncRNAs are preliminary and should be further substantiated with additional evidence or interpreted with more caution.

    We agree with the reviewer that we did not provide conclusive evidence that our phased ribosome footprinting data on mitochondrial non-coding RNAs are proof of novel translation events. We do acknowledge this in the main text:” Due to both the short ORFs, minimal read coverage, and lack of a detectable peptide we could not determine if translation elongation occurred on the mitochondrial tRNAs. These sites may be unproductive mitoribosome binding events or simply from tRNAs partially digesting during MNase treatment.”

    Reviewer #2 (Public review):

    In this study, the authors set out to explore how antibiotics known to inhibit bacterial protein synthesis also affect mitoribosomes in HEK cells. They achieved this through mitoribosome profiling, where RNase I and Mnase were used to generate mitoribosome-protected fragments, followed by sequencing to map the regions where translation arrest occurs. This profiling identified the codon-specific impact of antibiotics on mitochondrial translation.

    The study finds that most antibiotics tested inhibit mitochondrial translation similarly to their bacterial counterparts, except telithromycin, which exhibited distinct stalling patterns. Specifically, chloramphenicol and linezolid selectively inhibited translation when certain amino acids were in the penultimate position of the nascent peptide, which aligns with their known bacterial mechanism. Telithromycin stalls translation at an R/K-X-R/K motif in bacteria, and the study demonstrated a preference for arresting at an R/K/A-X-K motif in mitochondria. Additionally, alternative translation initiation sites were identified in MT-ND1 and MT-ND5, with non-canonical start codons. Overall, the paper presents a comprehensive analysis of antibiotics in the context of mitochondrial translation toxicity, and the identification of alternative translation initiation sites will provide valuable insights for researchers in the mitochondrial translation field.

    From my perspective as a structural biologist working on the human mitoribosome, I appreciate the use of mitoribosome profiling to explore off-target antibiotic effects and the discovery of alternative mitochondrial translation initiation sites. However, the description is somewhat limited by a focus on this single methodology. The authors could strengthen their discussion by incorporating structural approaches, which have contributed significantly to the field. For example, antibiotics such as paromomycin and linezolid have been modeled in the human mitoribosome (PMID: 25838379), while streptomycin has been resolved (10.7554/eLife.77460), and erythromycin was previously discussed (PMID: 24675956). The reason we can now describe off-target effects more meaningfully is due to the availability of fully modified human mitoribosome structures, including mitochondria-specific modifications and their roles in stabilizing the decoding center and binding ligands, mRNA, and tRNAs (10.1038/s41467-024-48163-x).

    These and other relevant studies should be acknowledged throughout the paper to provide additional context.

    We appreciate the work that has previously revealed how different antibiotics bind the mitochondrial ribosome. We have included these references in the manuscript to provide background and context for this work in relationship to the field.

    Reviewer #3 (Public review):

    Summary:

    Recently, the off-target activity of antibiotics on human mitoribosome has been paid more attention in the mitochondrial field. Hafner et al applied mitoribosome profilling to study the effect of antibiotics on protein translation in mitochondria as there are similarities between bacterial ribosome and mitoribosome. The authors conclude that some antibiotics act on mitochondrial translation initiation by the same mechanism as in bacteria. On the other hand, the authors showed that chloramphenicol, linezolid and telithromycin trap mitochondrial translation in a context-dependent manner. More interesting, during deep analysis of 5' end of ORF, the authors reported the alternative start codon for ND1 and ND5 proteins instead of previously known one. This is a novel finding in the field and it also provides another application of the technique to further study on mitochondrial translation.

    Strengths:

    This is the first study which applied mitoribosome profiling method to analyze mutiple antibiotics treatment cells.

    The mitoribosome profiling method had been optimized carefully and has been suggested to be a novel method to study translation events in mitochondria. The manuscript is constructive and written well.

    Weaknesses:

    This is a novel and interesting study, however, most of the conclusion comes from mitoribosome profiling analysis, as a result, the manuscript lacks the cellular biochemical data to provide more evidence and support the findings.

    We thank the reviewer for the positive assessment of our work. We agree that future biochemical and structural experiments will strengthen the conclusions we derive from the ribosome profiling.

    Recommendations for the authors:

    Reviewer #1 (Recommendations for the authors):

    In Fig. 1A, the quality of the Western blot for the sucrose gradient is suboptimal. I recommend enhancing the quality of the Western blot image and providing the sucrose gradient sedimentation patterns for both the mtSSU and mtLSU to confirm the accurate selection of the monosome fraction. Additionally, to correctly assign the A260 peaks to mitochondrial and cytosolic ribosomes, it would be helpful to include markers for both the cytoribosomal LSU and SSU, too. Furthermore, do the authors observe mitochondrial polysomes in their sucrose gradient? If so, were those fractions fully excluded from the analysis?

    We repeated our sucrose gradient and Western blotting with antibodies for the large and small subunits of the mitoribosome. We did not repeat western blotting for the cytoribosomes as the 40S, 60S, and 80S peaks are present in their canonical heights and locations on a sucrose gradient. Western blotting indicates that the large and small subunits of the mitoribosome are located in the fraction taken for mitoribo-seq. We do see trace amounts of mitoribosome in fractions past the 55S site. Those fractions were excluded from library preparation.

    The MNase footprints exhibited a bimodal distribution, which the authors suggest may indicate that "MNase-treatment may have captured two distinct conformations of the ribosome." It would be relevant to clarify whether an enzyme titration was performed, as excessive MNase could lead to ribosomal RNA degradation, potentially influencing the footprints.

    We did not perform a titration and instead based our concentration on the protocol from Rooijers et al, 2013. We included a statement of this and a reference to the concentration in the methods.

    Is there an explanation for why RNase I footprinting reveals a very high peak at the 5'-end of the MT-CYB transcript, whereas this is not observed with MNase footprinting?

    It is not clear. The intensity of peaks at the 5’ end of the transcripts varies. We do observe that the relative intensity of the 5’ peak is greater for RNase I footprinted samples than MNase-treated samples.

    I understand that throughout the manuscript, the authors use MT-CYB as an example to illustrate the effects of the antibiotics on mitochondrial translation. However, to strengthen the generality of the conclusions, it would be beneficial to provide the read distribution across the entire mitochondrial transcriptome, possibly in the supplementary material. Additionally, I suggest including the read distribution for MT-CYB in untreated cells to improve data interpretation and enhance the clarity of figures (e.g., Figs. 1B, 2B, 3B).

    As these experiments were generated across multiple mitoribo-seq experiments, each was done with its own control experiment. It would be inaccurate to show a single trace as representative of all experiments. Instead, we include Supplementary Figure 1, which shows the untreated MT-CYB trace for all control samples and indicates which treatment they pair with.

    It would be very valuable to label each individual data point in the read phasing shown in Fig. 1D with the corresponding transcripts. For improved data visualization, I suggest assigning distinct colors to each transcript.

    We are concerned that including the name of each gene in the main figure would be too difficult for the reader to accurately interpret. Instead, we have added a Supplementary Table with those values.

    How do the authors explain the significant peak (approx. 10,000 reads) at the 5' end of the transcript in the presence of tiamulin (Fig. 2B)? Does this peak correspond to the start codon, and how does it relate to the quantification reported in Fig. 2C?

    Yes, this represents the start codon. These reads are likely derived from the start codon as they are mapping to the 5’ end of the transcript. There are differences in sequencing depth depending on the experiment, so what is critical is the relative distribution of reads on the transcript rather than comparing absolute reads between experiments. MT-CYB has 54% of the reads at the start site, which is representative of what we see across all genes.

    Throughout the manuscript, I found the usage of the terms "5' end" and "start codon" somewhat confusing, as they appear to be used synonymously in some instances. For example, in Fig. 2C, the y-axis label states "ribosomes at start codon," while the figure caption mentions "...percentage of reads that map to the 5' end of mitochondrial transcripts." Given the size of the graphs, it is also challenging for the reader to determine whether the peaks correspond specifically to the start codon or if multiple peaks accumulate at the initial codons.

    We were selected for this language because two different types of analysis are being carried out. Ribosome profiling carried out in Figures 2 and 3 is carried out with RNase I, which poorly maps the ribosomes at the start codon when we do the read length analysis in Figure 4. Ribosome footprint at the 5’ end may include ribosomes that are on the 2-4 codons following the start codon, so it would not be accurate to label those as “ribosomes at a start codon.” We have renamed the axis to “Ribosomes at 5’ end”. Wig files are available online for all mitoribosome profiling experiments. In this case, the assigned “P-site” is several codons after the start codon due to the offset applied and the minimal 5’ UTR. Thus, it is less important to see which codon density is assigned to, but rather the general distribution of the reads.

    The authors state, "Cells treated with telithromycin did show a slight increase in MRPF abundance at the 5' end of MT-CYB" and "the cumulative distribution of MRPFs suggested that ribosome density was biased towards the 5' end of the gene for chloramphenicol and telithromycin, but not significantly for linezolid." Could this observation be linked to the presence of specific stalling motifs in that region? If so, it would be beneficial to display such motifs on the graphs of the read distribution across the transcriptome to substantiate the context-dependent inhibition.

    Thank you for this suggestion. For chloramphenicol and linezolid, alanine, serine, and threonine make up nearly 25% of the mitochondrial proteome. As such, there are numerous stall sites across the transcript. Given their identical stalling motifs, the difference between chloramphenicol and linezolid is due to sequence-specific differences. Potentially, this could be due to conditions such as the final concentration of antibiotic inside the mitochondria and the on/off rate of an inhibitor with the translating mitoribosome. Both may affect the kinetics of stalling and allow mitoribosomes to evade early stall sites.

    We have also included the sites of all A/K/R-X-K motifs located in the genome and the calculated fold change for each position. As a note, this includes sites that do not pass the minimum filter set by our analysis and we note this in the text.

    The comment raises an additional question: Does the increased density at the 5’ end derive from stalled mitoribosomes or queued mitoribosomes behind a stalling event? Recent work by Iwasaki’s group shows that mitoribosomes can form disomes and queue behind each other. However, we could not observe 30 aa periodicities behind stalling events that would be indicative of collided mitoribosomes.

    In Fig. 3E, the authors report an additional and very interesting observation that is not discussed. Linezolid treatment causes reduced ribosome occupancy when proline or glycine codons occupy the P-site, or when the amino acids have been incorporated into the polypeptide chain and occupy the -1 position. It is known that the translation of proline and glycine frequently leads to ribosome stalling due to the physicochemical properties of these amino acids. Has this effect of linezolid been reported in the bacterial translation system? Additionally, can the authors propose hypotheses for the mechanism behind this observation? A similar observation is noted for telithromycin when glycine occupies the same positions, as well as when aspartate occupies the P- and A-sites.

    In bacteria, Linezolid does have an “anti-stalling” motif when glycine is present in the A-site. However, this is due to the size of the residue being compatible with antibiotic binding.

    The most likely cause of this effect is a redistribution of ribosome footprints. As the antibiotics introduce new arrest sites, ribosome density at other sites relatively decreases. This is likely an artifact from mitoribosomes redistributing from endogenously slow codons to new arrest sites. When looking at carrying out our disome profiling in the presence of anisomycin, we see a similar effect. Cytoribosomes are redistributed from endogenous stalling sites, such as proline, and are redistributed throughout the gene. As a result, translation at proline appears “more efficient” upon treatment with an inhibitor but is instead an artifact of analysis.

    Figure 3F could benefit from indicating which mtDNA-encoded protein corresponds to each of the strongest stalling motifs.

    We have included a supplementary figure to highlight which mitochondrially-encoded genes containing the R/K/A-X-K motif and noted in the text that mitochondrial translation may be unevenly inhibited.

    The legend "increasing mRPF abundance" in Fig. 4C may be missing the corresponding colors.

    The legend applies to all sections of the figure. We double-checked the range of the colors in the tables, and they do match the legend.

    The observation that the start codons in MT-ND1 and MT-ND5 might differ from the annotated canonical ones is intriguing. While the ribosome profiling data appear clear, mass spectrometry (MS) analysis may be misleading. The absence of evidence does not necessarily imply evidence of absence. How does this proposed conclusion correlate with the structural data obtained from HEK cells? For instance, the cryo-EM structural model of a complex I-containing human supercomplex (PDB: 5XTD, PMID: 28844695) shows the presence of Pro2 in MT-ND1 and the full-length MT-ND5 protein. The authors should carefully examine structural data to ascertain whether alternative forms of MT-ND1 and MT-ND5 are actually observed in the assembled complex I.

    We really appreciate this comment. We sat down with an expert in cryo-EM and reviewed the figure. We downloaded the density map and reviewed the N-termini of MT-ND1 and MT-ND5. We only observed the density of the N-terminus of MT-ND1 at low confidence. At an RMSD of 2, we could not observe density for the side chains of Met and Pro, and there are gaps in the density for what is modeled as the main chain. The assignment of these residues may have been overlooked due to the expectation that they should be present in the peptide.

    For MT-ND5, we did observe some density that could be part of the main chain; however, it did not fill out until we reduced the stringency, and we did not observe density mapping to side chain residues. To summarize, we do not confidently see density for either the side chain or the main chain for either peptide.

    Given that ribosome profiling is based on the assumption that ribosomes protect mRNA fragments from RNase digestion, interpreting the data related to Fig. 5 and the proposed existence of translation events involving ncRNAs is challenging. Most importantly, tRNAs and rRNAs are highly folded RNA molecules and, by definition, are protected by ribosomal proteins. Simultaneously, as the authors point out, "These reads could either be products of random digestion of the abundant background of ncRNAs or be genuine MRPFs." RNase I preferentially digests single-stranded RNA (ssRNA), but excess enzyme can still lead to degradation. Consequently, many random tRNA/rRNA fragments may be generated by RNase digestion, potentially resulting in artifacts. I suggest that the authors examine what happens to these reads when mitochondrial translation is inhibited.

    We have low-quality mitoribo-seq with initiation inhibitors and Mnase showing footprints of the same size. We do not have a small-molecule inhibitor that is able to completely ablate translation, as they instead stabilize mitoribosomes at different steps in translation. We have considered alternative ways of capturing a background rRNA and tRNA digestion pattern; however, these have their own drawbacks. Dissociation with EDTA prior to digestion or carrying out library prep on the small and large subunits may capture mitoribosomes no longer in the process of translation; however, dissociated subunits would have different surfaces now available for digestion and may not capture tRNAs.

    Regarding the statement, "While the ORF on MT-TS1 is longer, MRPF density was low and we did not observe read phasing and thus it is likely not translated (not shown)," the data should not be excluded unless a clear explanation is provided for why translation would not occur from this specific RNA.

    We have included this value in the graph as well as in Supplementary Figure 1.

    The graph in Fig. 5B shows the periodicity of only the putative RNR1 ORF, but not that of the other proposed ORFs. What is the reason for this?

    We have included the MT-TS1 putative ORF in Figure 5 and Figure S1. Other ORFs did not have density in the ORF. If these are real mitoribosome footprints at these start codons, it may be due to them being transient binding events that never result in elongation. Alternatively, they may be due to tRNA degradation during library preparation.

    The assumption that the UUG codon can serve as a start site for mitochondrial translation has not been substantiated. Recent data have identified translation initiation events from non-ATG/ATA codons (near-cognate and sub-cognate) using retapamulin, but UUG was not among them. Can the authors detect such events in their ribosome profiling data collected in the presence of retapamulin, tiamulin, or josamycin?

    The report of translation initiation at non-ATG/ATA codons strongly disagrees with our findings. We report that sites of translation initiation observed within annotated coding regions in mitochondria occur at the annotated start sites, while the other report finds frequent alternative initiation events. We have looked for those arrest sites and did not observe them.

    In the section "Mitoribosome profiling reveals novel translation events," the title may be misleading given the preliminary nature of the results. To support such a claim, the authors should provide experimental evidence demonstrating that the proposed translation events genuinely exist and result in the synthesis of previously unidentified polypeptides. Alternatively, the interpretation should be approached with greater caution and more clearly indicated as preliminary.

    We agree with the reviewers that a distinction should be made between reporting truly novel translation events, like the recently reported MT-ND5-dORF, and sites we suspect mitoribosomes may be binding and that require detailed follow-up. We altered the section title to suggest that this may be showing novel translation events. Additionally, we included a statement in the discussion that these MRPFs may be simply tRNA digestion by RNase I.

    Although located at the 5' end of RNR1, the newly identified ORF is situated 79 nt downstream. According to current knowledge, this appears to be a lengthened 5' UTR that may hinder mitoribosome loading. The authors should speculate on potential initiation mechanisms.

    The start of the putative ORF is not located 79 nts down, but at the 8th nucleotide. The reviewer may be including the tRNA-Phe in their calculation, which is cleaved from MT-RNR1. This start site is closer to the 5’ end than our findings with MT-ND5.

    To enhance the interpretation of the mitoribosome profiling data, the authors could complement their findings with classical metabolic labeling using (35)S-methionine. This approach would allow for a different assessment of the stringency of the inhibition under the tested experimental conditions.

    We are currently working on these experiments using mito-funcats. A future direction we are taking this work is to understand how the cell responds to different mechanisms of translation inhibition. For example, we are trying to understand if telithromycin, which appears highly selective, only partially inhibits translation of the mtDNA-encoded proteome.

    Reviewer #2 (Recommendations for the authors):

    Other small editorial comments:

    Line 24: "translate proteins"?

    Revised for clarity

    Line 24: The sentence describing mitochondrial translation as "closely resembling the one in prokaryotes" could be reformulated. While the core of the mitoribosome is conserved, the entire apparatus has many mitochondria-specific features.

    Since this is the abstract, we simplified the point by saying that mitoribosomes are more similar to prokaryotic than cytosolic ribosomes.

    Clarified to highlight that the mitochondrial system is more similar to the bacterial system than the eukaryotic system.

    Line 33: "novel" or "previously unrecognized" ?

    Rewritten for clarity.

    Lines 33-35: The claim made here is not shown in the paper.

    We removed the more aspirational goal of this paper and focused on the main findings of the paper.

    Lines 44, 47, 89 (and elsewhere): "cytoplasmic" or "cytosolic" ?

    Both terms are used in the literature. We opted for cytoplasmic as it can also include ribosomes not free in the cytosol, such as those bound to the ER.

    Reviewer #3 (Recommendations for the authors):

    (1) The authors should state why they chose these antibiotics for mitoribosome profiling analysis over other antibiotics from same group. Did they screen multiple antibiotics to determine the candidates for next step?

    We selected antibiotics that had a known stalling motif in bacteria (initiation or context-dependent elongation inhibitors). In addition, we carried out mitoribosome profiling with erythromycin, azithromycin, thiostrepton, and kanamycin in this work. However, we did not see any effect from these drugs in mitoribosome profiling. We are currently testing other inhibitors, such as doxycycline and tigecycline, and looking at optimizing treatment conditions to identify stalling motifs in samples that previously showed no difference.

    (2) What is the reason for choosing the concentration of antibiotics retapamulin, tiamulin and josamycin, this is IC50 value or above this value? On the other hand, none of this information has been provided for the antibiotics in the next part. The authors should provide biochemical study for the effect of these antibiotics on cell survival and/or protein translation such as S35 assay or steady state level of mtDNA-encoded proteins upon cell treatment with these antibiotics.

    Prior to mitoribo-seq, we carried out time and concentration assays with all antibiotics. 100 µg/ml and a 30-minute treatment was tolerable for all antibiotics except retapamulin. We aimed to treat cells with a relatively high concentration of inhibitor in order to capture actively translating mitoribosomes. We were concerned that longer treatments may lead to decreased translation initiation, leading to the capture of fewer mitoribosomes. These concentrations were nearly identical to contemporary conditions carried out in Bibel et al, RNA 2025.

    (3) Why did the authors choose MT-CYB as the representative for further analysis in the second and third parts of the manuscript?

    We chose MT-CYB because its length allowed for easy visualization. Some mitochondrial genes, such as MT-ND6, had a propensity for stronger stalling at initiation. While coverage was throughout the genes, it was difficult to visualize the changes within the ORF. Also, MT-CYB was less visually complex than polycistronic transcripts. All wigs were uploaded to GEO.

    (4) Page 11, line 233-234: the authors state that telithromycin induces stalling at R/K/A-X-K motif. The authors should do further analysis on mitochondrial genome which proteins contain this motif. Furthermore, same as comment 2: the authors should confirm by 35S assay or WB to know which mtDNA-encoded proteins are affected.

    We have included a supplementary figure showing which mitochondrial genes contain these motifs.

    (5) The results and conclusion from the fourth paragraph are very interesting. The authors suggest alternative start codon for two mtDNA encoded proteins: ND1 and ND5 based on ribosome profiling analysis. Again, I have several comments on this part:
    (a) For the accumulation of the alternative start codon of ND1 and ND5 as suggested in the manuscript, do the authors observe this trend with the initiation inhibitors used in the second paragraphs of the manuscript?

    We did not observe similar read lengths with retapamulin, tiamulin, or josamycin, which produced read lengths that were consistent with other RNase I footprinted samples.

    (b) This observation was further confirmed by MS with a peptide form ND1 protein, the authors should show MS peak indicating MW of the peptide and MS/MS data for the peptide which supports this hypothesis.

    We are including the MS/MS report for this peptide.

    (c) Interestingly, several high-resolution structures of mammalian complex I have been reported so far (PMID: 7614227, 10396290, 38870289), ND1 and ND5 protein express full sequences with fMet at the distal N-terminal. This is different to the suggestion from the manuscript. Could the author discuss or comment on that?

    This point was brought up by another reviewer. We have carefully analyzed the density map of PMID: 28844695. We sat down with an expert in cryo-EM and reviewed the figure. We downloaded the density map and reviewed the N-termini of MT-ND1 and MT-ND5. We only observed the density of the N-terminus of MT-ND1 at low confidence. At an RMSD of 2, we could not observe density for the sidechains of Met and Pro, and there is a gap in density for what is modeled as the main chain. The assignment of these residues may have been overlooked due to the expectation that they should be present in the peptide.

    For MT-ND5, we did observe some density that could be part of the main chain; however, it did not fill out until we reduced the stringency, and we did not observe density mapping to side chain residues. To summarize, we do not confidently see density for either the side chain or the main chain for either peptide.

    Minor comments:

    The method should be written more accurately for easily repeating experiments by other groups. For example:

    (1) The authors should indicate what was exact HEK293 cell line used in this study.

    We have indicated the exact cell line.

    (2) Page 22, line 471: which (number) fractions had been collected. The Western Blot analysis shown in Figure 1A should be repeated with both proteins from small and large subunits.

    We have repeated the Western blot with antibodies for large and small subunits. We took fractions 8 and 9, which are now indicated in the text and figure.

    (3) Page 23, line 502: is this number of cells used for MS experiment is correct? Or is this number of cells per mL?

    This is correct and is based on the kit protocol. It is not cells per mL. We have clarified the kit being used in the methods.

  5. eLife

    eLife Assessment

    This useful study presents findings on how some antibiotics, which inhibit protein synthesis in bacteria, affect the translation in mitochondrial ribosomes. The authors provide solid evidence that most tested antibiotics act similarly on bacterial and mitochondrial translation. Additionally, this work shows that alternative translation initiation events might exist in two specific mt-mRNAs (MT-ND1 and MT-ND5). The conclusions of this manuscript are of broad interest to the antibiotic and the mitochondrial fields.

  6. eLife

    Reviewer #1 (Public review):

    Summary:

    This study aimed to determine whether bacterial translation inhibitors affect mitochondria through the same mechanisms. Using mitoribosome profiling, the authors found that most antibiotics, except telithromycin, act similarly in both systems. These insights could help in the development of antibiotics with reduced mitochondrial toxicity.
    They also identified potential novel mitochondrial translation events, proposing new initiation sites for MT-ND1 and MT-ND5. These insights not only challenge existing annotations but also open new avenues for research on mitochondrial function.

    Strengths:

    Ribosome profiling is a state-of-the-art method for monitoring the translatome at very high resolution. Using mitoribosome profiling, the authors convincingly demonstrate that most of the analyzed antibiotics act in the same way on both bacterial and mitochondrial ribosomes, except for telithromycin. Additionally, the authors report possible alternative translation events, raising new questions about the mechanisms behind mitochondrial initiation and start codon recognition in mammals.

    Weaknesses:

    The main weaknesses of this study are:
    - While the authors highlight an interesting difference in the inhibitory mechanism of telithromycin on bacterial and mitochondrial ribosomes, mechanistic explanations or hypotheses are lacking.
    - The assignment of alternative start codons in MT-ND1 and MT-ND5 is very interesting but does not seem to fully align with structural data.
    - The newly proposed translation events in the ncRNAs are preliminary and should be further substantiated with additional evidence or interpreted with more caution.

  7. eLife

    Reviewer #2 (Public review):

    In this study, the authors set out to explore how antibiotics known to inhibit bacterial protein synthesis also affect mitoribosomes in HEK cells. They achieved this through mitoribosome profiling, where RNase I and Mnase were used to generate mitoribosome-protected fragments, followed by sequencing to map the regions where translation arrest occurs. This profiling identified the codon-specific impact of antibiotics on mitochondrial translation.

    The study finds that most antibiotics tested inhibit mitochondrial translation similarly to their bacterial counterparts, except telithromycin, which exhibited distinct stalling patterns. Specifically, chloramphenicol and linezolid selectively inhibited translation when certain amino acids were in the penultimate position of the nascent peptide, which aligns with their known bacterial mechanism. Telithromycin stalls translation at an R/K-X-R/K motif in bacteria, and the study demonstrated a preference for arresting at an R/K/A-X-K motif in mitochondria. Additionally, alternative translation initiation sites were identified in MT-ND1 and MT-ND5, with non-canonical start codons. Overall, the paper presents a comprehensive analysis of antibiotics in the context of mitochondrial translation toxicity, and the identification of alternative translation initiation sites will provide valuable insights for researchers in the mitochondrial translation field.

    From my perspective as a structural biologist working on the human mitoribosome, I appreciate the use of mitoribosome profiling to explore off-target antibiotic effects and the discovery of alternative mitochondrial translation initiation sites. However, the description is somewhat limited by a focus on this single methodology. The authors could strengthen their discussion by incorporating structural approaches, which have contributed significantly to the field. For example, antibiotics such as paromomycin and linezolid have been modeled in the human mitoribosome (PMID: 25838379), while streptomycin has been resolved (10.7554/eLife.77460), and erythromycin was previously discussed (PMID: 24675956). The reason we can now describe off-target effects more meaningfully is due to the availability of fully modified human mitoribosome structures, including mitochondria-specific modifications and their roles in stabilizing the decoding center and binding ligands, mRNA, and tRNAs (10.1038/s41467-024-48163-x).
    These and other relevant studies should be acknowledged throughout the paper to provide additional context.

  8. eLife

    Reviewer #3 (Public review):

    Summary:

    Recently, the off-target activity of antibiotics on human mitoribosome has been paid more attention in the mitochondrial field. Hafner et al applied mitoribosome profilling to study the effect of antibiotics on protein translation in mitochondria as there are similarities between bacterial ribosome and mitoribosome. The authors conclude that some antibiotics act on mitochondrial translation initiation by the same mechanism as in bacteria. On the other hand, the authors showed that chloramphenicol, linezolid and telithromycin trap mitochondrial translation in a context-dependent manner. More interesting, during deep analysis of 5' end of ORF, the authors reported the alternative start codon for ND1 and ND5 proteins instead of previously known one. This is a novel finding in the field and it also provides another application of the technique to further study on mitochondrial translation.

    Strengths:

    This is the first study which applied mitoribosome profiling method to analyze mutiple antibiotics treatment cells.
    The mitoribosome profiling method had been optimized carefully and has been suggested to be a novel method to study translation events in mitochondria. The manuscript is constructive and written well.

    Weaknesses:

    This is a novel and interesting study, however, most of the conclusion comes from mitoribosome profiling analysis, as a result, the manuscript lacks the cellular biochemical data to provide more evidence and support the findings.

  9. Version published to 10.7554/elife.103699.1 on eLife
  10. Version published to 10.7554/elife.103699 on eLife
  11. Version published to 10.1101/2024.08.29.610400 on bioRxiv