A tRNA processing enzyme is a key regulator of the mitochondrial unfolded protein response

  1. James P Held
  2. Gaomin Feng
  3. Benjamin R Saunders
  4. Claudia V Pereira
  5. Kristopher Burkewitz
  6. Maulik R Patel

  • Curated by eLife

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    Evaluation Summary:

    This manuscript reports that tRNA processing enzyme HOE-1 is required for the activation of mitochondrial unfolded protein response in C. elegans. This study extends our understanding of how the mitochondria-nuclear communication is mediated via a tRNA processing enzyme, and can serve as a staring point to elucidate the mechanism by which HOE-1 regulates mitochondrial unfolded protein response.

    (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. The reviewers remained anonymous to the authors.)

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Abstract

The mitochondrial unfolded protein response (UPR mt ) has emerged as a predominant mechanism that preserves mitochondrial function. Consequently, multiple pathways likely exist to modulate UPR mt . We discovered that the tRNA processing enzyme, homolog of ELAC2 (HOE-1), is key to UPR mt regulation in Caenorhabditis elegans . We find that nuclear HOE-1 is necessary and sufficient to robustly activate UPR mt . We show that HOE-1 acts via transcription factors ATFS-1 and DVE-1 that are crucial for UPR mt . Mechanistically, we show that HOE-1 likely mediates its effects via tRNAs, as blocking tRNA export prevents HOE-1-induced UPR mt . Interestingly, we find that HOE-1 does not act via the integrated stress response, which can be activated by uncharged tRNAs, pointing toward its reliance on a new mechanism. Finally, we show that the subcellular localization of HOE-1 is responsive to mitochondrial stress and is subject to negative regulation via ATFS-1. Together, we have discovered a novel RNA-based cellular pathway that modulates UPR mt .

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  1. Version published to 10.7554/elife.71634 on eLife
  2. eLife

    Author Response:

    Reviewer #1 (Public Review):

    In this manuscript, the authors identified HOE-1, a tRNA processing enzyme, as an important regulator of UPRmt. They showed that nuclear HOE-1 is necessary and sufficient to activate UPRmt, which acts through ATFS-1 and DVE-1. The authors provided evidence that the UPRmt induced by nuclear retention of HOE-1 requires 3'-tRNA processing and tRNA transport. Moreover, HOE-1 is negatively regulated by ATFS-1 when UPRmt is activated. The experiments were well executed, and data are clear and convincing.

    Comments:

    1. The authors showed that HOE-1 localized to both mitochondria and nucleus in germline, while HOE-1(ΔNES) induces UPRmt in the intestine. The HOE-1 localization in the intestine should be presented including mitochondria and nucleus. The authors suggested that HOE-1 activates UPRmt in the intestine in a cell-autonomous manner. This would need to be demonstrated experimentally.

    We agree that both points here are important based on the conclusions we draw in the manuscript. We have experimentally addressed both points by conducting high resolution imaging of HOE1::GFP in the germline (with mitochondrial co-marker TMRE) and tissue-specific knockdown of HOE-1 protein.

    1. Whether 3'-tRNA processing is elevated in HOE-1(ΔNES) should be tested more directly. Is it possible to do determine the tRNA species that are elevated in HOE-1(ΔNES) strain by sequencing? Or that the authors can express HOE-1(ΔNES) that lacks the enzymatic activity and see whether it can still activate UPRmt.

    This is an important point. Thus we took multiple approaches to test the function of HOE-1(ΔNES) including assessing nuclear levels of HOE-1(ΔNES) vs wildtype HOE-1, nuclear requirement of HOE-1(ΔNES), for UPRmt activation, and dependence upon enzymatic activity as suggested.

    1. The images shown in Fig. 8a is not clear. Enlarged images would be needed to clearly show changes in HOE-1 subcellular localization (mitochondria and nucleus) upon multiple mitochondria stresses.

    We conducted high resolution confocal microscopy to complement our whole animal imaging allowing us to assess more thoroughly changes in sub-cellular localization of HOE-1 in mitochondrial stressed (nuo-6(qm200) animals and in animals with constitutive UPRmt activation (atfs-1(et15)).

    1. The elevation of HOE-1 protein level is not clear (Fig. 8c, d). It is unclear whether the HOE-1 level in nuo-6 with atfs-1RNAi issignificantly increased compared with control RNAi or that in wild type with atfs-1 RNAi. The HOE-1 intensity in mitochondria vs nucleus would need to be examined in multiple mito stress conditions. It is also unclear how HOE-1 senses Mito stress.

    We have addressed all of the reviewers comments listed here. First, to more thoroughly demonstrate increased HOE-1 protein levels during mitochondrial stress in the absence of atfs-1 we assessed HOE-1::GFP protein levels in additional biological replicates and found that HOE- 1::GFP is significantly increased during mitochondrial stress in the absence of atfs-1. Second, we assessed HOE-1 nuclear dynamics under two additional mitochondrial stressors and found that HOE-1 nuclear levels are depleted in those conditions as well. Finally, we addressed how it may be that HOE-1 senses mitochondrial stress in the discussion section.

    Reviewer #2 (Public Review):

    This manuscript reports that the tRNA processing enzyme HOE-1 is required for the activation of UPRmt in C. elegans. Given the dual-localization of HOE-1, the authors create mitochondrial and nuclear compartment-specific knockout of HOE-1 and demonstrated that only the nuclear HOE-1 is necessary and sufficient to activate the UPRmt. This paper will be of interest to scientists within mitochondrial stress response signaling. This study extends our understanding of how the mito-nuclear communication is mediated via the tRNA processing enzyme. However, some key aspects of the study need to be reinforced in the conclusions.

    1. The phenotype that HOE-1(NLS) mutants suppressed the induction of the UPRmt in mitochondrial mutants is interesting. However, the mechanism underlying the HOE-1-mediated mitochondrial stress response is still not very clear. I have concerns regarding the specific involvement of HOE-1 in the regulation of UPRmt, since tRNA processing, the tRNA exporter xpo-3, as well as the RNase P complex popl-1, are all general regulators for protein synthesis. It is unclear how one can explain the specific involvement of these regulators only in the regulation of the UPRmt.

    The reviewer raises an important point. Indeed these enzymes have known essential functions. However, our data suggests that they also play an important role in UPRmt regulation specifically. We reason that hoe-1(ΔNES)-induced UPRmt is more sensitive to changes in tRNA regulation than protein translation. This reasoning is supported by our finding that animals on xpo-3 and popl-1 RNAi develop like wildtype.

    1. It is also confusing that HOE-1(NLS) mutants suppressed the UPRmt induction in nuo-6 mutants, however, xpo-3 which functions in the same pathway as HOE-1 in terms of tRNA processing and export did not suppress the UPRmt induction in nuo-6 mutants in Fig 6i and 6j.

    We agree the differential impact of xpo-3 RNAi on hoe-1(ΔNES)- and nuo-6(qm200)-induced UPRmt is interesting. While HOE-1 processed tRNAs play a role in activating UPRmt in response to mitochondrial stress, ATFS-1 is also capable of activating UPRmt directly. In contrast, HOE-1 processed tRNAs are presumably solely responsible for UPRmt activation in hoe-1(ΔNES) animals and hence completely dependent on their exporter XPO-3.

    1. The authors mentioned that HOE-1 homolog ELAC2 is not only required for tRNA maturation but also essential for the formation of tRNA fragments, snoRNAs, and miRNAs, are these non-coding RNAs account for the activation of the UPRmt?

    It is possible that RNA(s) aside from tRNAs whose maturation is hoe-1-dependent could be involved in UPRmt regulation. We address this possibility in the discussion and look forward to identifying the causal RNA in future studies.

    1. It is interesting to show that hoe-1(ΔNES) mutant is sufficient to induce the nuclear accumulation of the ATFS-1 and the subsequent up-regulation of the UPRmt reporter gene. However, the authors did not rule out the possibility that mitochondrial protein homeostasis was already disrupted in hoe-1(ΔNES) mutants so that the UPRmt was induced.

    We thank the reviewer for this important point. We conducted TMRE staining on wildtype and hoe-1(ΔNES) adult animals and find that mitochondrial membrane potential is drastically decreased in hoe-1(ΔNES) adults. This membrane potential depletion is not atfs-1-dependent suggesting that hoe-1(ΔNES) directly compromises mitochondrial membrane potential.

    1. The authors only showed that mitochondrial membrane potential was not changed in hoe-1(ΔNES) mutants. More characterization of mitochondrial function in hoe-1(ΔNES) mutants is required, such as OCR and mitochondrial morphology. It seems that hoe-1(ΔNES) mutants are smaller than wild-type animals.

    In response to these reviews we more thoroughly assessed mitochondrial membrane potential in hoe-1(ΔNES) adults. High resolution microscopy reveals that mitochondrial membrane potential is depleted in hoe-1(ΔNES) adult animals.

    1. In fig 4a, Why the overall level of ATFS-1 is dramatically increased in hoe-1(ΔNES) mutants, this is not consistent with only two-fold up-regulation of atfs-1 transcript levels. The authors also would need to show the ATFS-1::GFP expression pattern in the nuo-6 mutants as a control.

    In response to this review, thorough assessment of ATFS-1::mCherry nuclear localization and total protein levels by high resolution confocal microscopy suggest that total cellular ATFS-1 levels are not elevated in hoe-1(ΔNES) animals relative to wildtype. As suggested, we also assessed nuclear and total ATFS-1 levels in nuo-6(qm200) animals as a positive control.

    Reviewer #3 (Public Review):

    Held and colleagues present numerous intriguing findings suggesting that the tRNA processing enzyme ELAC2/HOE-1 is required to activate the mtUPR in C. elegans. The hoe-1 gene encodes 2 proteins, one of which contains a mitochondrial targeting sequence (MTS) and a nuclear localization sequence (NLS). The other protein is similar but lacks the MTS. Thus, hoe-1 encodes proteins involved in tRNA processing in the nucleus and within mitochondria. It is intriguing that one or both of the proteins may be required for mtUPR activation. I have multiple concerns related to the experimental design and the interpretation. However, my major concern is that it remains unclear how HOE-1 regulates the mtUPR (DVE-1 or ATFS-1).

    Major concerns.

    -Figure 1. The authors use the transcriptional reporter hsp-6::gfp as a mtUPR reporter. However, in addition to requiring transcription for the hsp-6 promoter to induce the gfp mRNA, that mRNA must be synthesized. As HOE-1 is a tRNA processing enzyme likely required for protein synthesis, qRT-PCR analysis should be performed to quantify the effects of HOE-1 inhibition on the mtUPR transcription response. Thus the data supporting the claim that loss-of-function mutations in HOE-1 inhibit mtUPR-dependent transcription are weak and must be further substantiated.

    We thank the reviewer for raising this point. As recommended, we performed quantitative PCR (i.e. droplet digital PCR) to quantify transcripts of genes upregulated upon UPRmt activation (i.e. hsp-6 and cyp-14A4.1) in a wildtype and hoe-1(ΔNLS) background in the absence and presence of mitochondrial stress (control and spg-7 RNAi, respectively) and find that UPRmt transcripts are highly reduced in hoe-1(ΔNLS) during mitochondrial stress.

    • Several groups have shown that inhibition of S6 kinase inhibits mtUPR activation. As HOE-1 is presumably required for protein synthesis, perhaps the mechanism is related? It would be good to know whether the inhibition of other genes affecting tRNA levels also impairs mtUPR or is specific to HOE-1.

    To address the reviewer’s point we assessed if inhibition of other genes affecting tRNA levels also impair UPRmt in addition to hoe-1 and popl-1 which we show in the original manuscript. We tested the effects of RNA polymerase III dependent transcription by RNAi against pol III subunit rpc-1, as well as other downstream tRNA maturation steps including tRNA ligation (rtcb-1 RNAi) and CCA-addition (hpo-31 RNAi) on UPRmt activation.

    -It is my understanding that the HOE-1 protein with a mitochondrial targeting sequence is transcribed from the same gene as HOE-1 without the MTS. And, there are separate transcriptional start sites for each mRNA/protein. Considering the number of claims related to subcellular localization of HOE-1, the authors would need to determine if transcription from either site is altered during mitochondrial stress.

    We assessed transcription from the hoe-1 gene locus under conditions of mitochondrial stress relative to no stress using primers specific for mitochondrial targeted HOE-1 and total hoe-1 transcripts. We found that total hoe-1 transcript levels are elevated under conditions of stress. However, we find no difference in transcript level between mitochondrial-specific and total hoe-1 suggesting that there is only one transcript for hoe-1 that is used for translation of both mitochondrial and nuclear targeted HOE-1 protein. This is consistent with how the ortholog of hoe-1 in mammalian systems is regulated.

    • There is an over-reliance on the hoe-1(∆NES) strain which causes mtUPR activation. It remains unclear if nuclear accumulation is an event driving mtUPR activation or if the activation is simply an artifact of the ∆NES mutation. The hoe-1 loss of function studies are need to be further developed in order to interpret the hoe-1(∆NES) results. It remains possible that the ∆NES findings are simply an artifact of a neomorphic allele and do not inform on HOE-1 function.

    We appreciate this point of concern from the reviewer. We took four independent approaches to further characterize the cellular role of hoe-1(∆NES). We show that HOE-1 enzymatic activity is required for UPRmt activation by hoe-1(∆NES). We show that there are elevated nuclear levels of HOE-1 in hoe-1(∆NES) animals by high resolution microscopy supporting our hypothesis that increased levels of tRNA processing in the nucleus drives UPRmt activation. We show that HOE1 is required in the nucleus to activate UPRmt as UPRmt is turned off in hoe-1(∆NLS+∆NES) animals. Finally, we show that loss of HOE-1 from mitochondria in hoe-1(∆NES) containing animals (hoe-1(∆MTS+∆NES)) does not compromise UPRmt activation ruling out the possibility that hoe-1(∆NES) confers a neomorphic function in mitochondria to activate UPRmt.

    -The data suggesting that nuclear accumulation of HOE-1 is sufficient to activate mtUPR is relatively weak. Does HOE-1∆NES cause mitochondrial dysfunction which increases mtUPR activation? Potentially, HOE-1 lacking the nuclear export sequence may not accumulate within mitochondria and cause mitochondrial dysfunction. More in depth quantitative assessment of mitochondrial activity is required (TMRE images, oxygen consumption, etc). Alternatively, the ∆NES mutation could be combined with the ∆MTS mutation.

    To address this point we show that hoe-1(∆NES) causes mitochondrial membrane potential depletion by TMRE staining providing a mechanism by which hoe-1(∆NES) causes ATFS-1 nuclear accumulation and subsequent UPRmt activation. We show that in hoe-1(∆MTS+∆NES) animals UPRmt is still activated, ruling out hoe-1(∆NES) causing UPRmt activation by functioning in the mitochondria.

    -Fig 4. The authors generate a beautiful ATFS-1::mCherry fusion protein and demonstrate that accumulates within nuclei during mitochondrial stress. Does hoe-1 inhibition affect translation/synthesis of ATFS-1::mCherry or nuclear accumulation of ATFS-1::mCherry? Or, DVE-1?

    To directly address the impact of HOE-1 on ATFS-1 and DVE-1 protein levels we assessed total cellular ATFS-1::mCherry levels by confocal microscopy and total cellular DVE-1::GFP levels by western blot. We find that HOE-1 has no significant effect on total accumulation suggesting that HOE-1 drives UPRmt by increasing nuclear accumulation of both transcription factors.

    The mechanism by which hoe-1 impacts mtUPR is unclear.

    The experiments we conducted to thoroughly assess the role of HOE-1 in UPRmt activation provide deeper understanding and characterization of hoe-1-dependent UPRmt.

  3. eLife

    Evaluation Summary:

    This manuscript reports that tRNA processing enzyme HOE-1 is required for the activation of mitochondrial unfolded protein response in C. elegans. This study extends our understanding of how the mitochondria-nuclear communication is mediated via a tRNA processing enzyme, and can serve as a staring point to elucidate the mechanism by which HOE-1 regulates mitochondrial unfolded protein response.

    (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. The reviewers remained anonymous to the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    In this manuscript, the authors identified HOE-1, a tRNA processing enzyme, as an important regulator of UPRmt. They showed that nuclear HOE-1 is necessary and sufficient to activate UPRmt, which acts through ATFS-1 and DVE-1. The authors provided evidence that the UPRmt induced by nuclear retention of HOE-1 requires 3'-tRNA processing and tRNA transport. Moreover, HOE-1 is negatively regulated by ATFS-1 when UPRmt is activated. The experiments were well executed, and data are clear and convincing.

    Comments:

    1. The authors showed that HOE-1 localized to both mitochondria and nucleus in germline, while HOE-1(ΔNES) induces UPRmt in the intestine. The HOE-1 localization in the intestine should be presented including mitochondria and nucleus. The authors suggested that HOE-1 activates UPRmt in the intestine in a cell-autonomous manner. This would need to be demonstrated experimentally.

    2. Whether 3'-tRNA processing is elevated in HOE-1(ΔNES) should be tested more directly. Is it possible to do determine the tRNA species that are elevated in HOE-1(ΔNES) strain by sequencing? Or that the authors can express HOE-1(ΔNES) that lacks the enzymatic activity and see whether it can still activate UPRmt.

    3. The images shown in Fig. 8a is not clear. Enlarged images would be needed to clearly show changes in HOE-1 subcellular localization (mitochondria and nucleus) upon multiple mitochondria stresses.

    4. The elevation of HOE-1 protein level is not clear (Fig. 8c, d). It is unclear whether the HOE-1 level in nuo-6 with atfs-1RNAi issignificantly increased compared with control RNAi or that in wild type with atfs-1 RNAi. The HOE-1 intensity in mitochondria vs nucleus would need to be examined in multiple mito stress conditions. It is also unclear how HOE-1 senses Mito stress.

  5. eLife

    Reviewer #2 (Public Review):

    This manuscript reports that the tRNA processing enzyme HOE-1 is required for the activation of UPRmt in C. elegans. Given the dual-localization of HOE-1, the authors create mitochondrial and nuclear compartment-specific knockout of HOE-1 and demonstrated that only the nuclear HOE-1 is necessary and sufficient to activate the UPRmt. This paper will be of interest to scientists within mitochondrial stress response signaling. This study extends our understanding of how the mito-nuclear communication is mediated via the tRNA processing enzyme. However, some key aspects of the study need to be reinforced in the conclusions.

    1. The phenotype that HOE-1(NLS) mutants suppressed the induction of the UPRmt in mitochondrial mutants is interesting. However, the mechanism underlying the HOE-1-mediated mitochondrial stress response is still not very clear. I have concerns regarding the specific involvement of HOE-1 in the regulation of UPRmt, since tRNA processing, the tRNA exporter xpo-3, as well as the RNase P complex popl-1, are all general regulators for protein synthesis. It is unclear how one can explain the specific involvement of these regulators only in the regulation of the UPRmt.

    2. It is also confusing that HOE-1(NLS) mutants suppressed the UPRmt induction in nuo-6 mutants, however, xpo-3 which functions in the same pathway as HOE-1 in terms of tRNA processing and export did not suppress the UPRmt induction in nuo-6 mutants in Fig 6i and 6j.

    3. The authors mentioned that HOE-1 homolog ELAC2 is not only required for tRNA maturation but also essential for the formation of tRNA fragments, snoRNAs, and miRNAs, are these non-coding RNAs account for the activation of the UPRmt?

    4. It is interesting to show that hoe-1(ΔNES) mutant is sufficient to induce the nuclear accumulation of the ATFS-1 and the subsequent up-regulation of the UPRmt reporter gene. However, the authors did not rule out the possibility that mitochondrial protein homeostasis was already disrupted in hoe-1(ΔNES) mutants so that the UPRmt was induced.

    5. The authors only showed that mitochondrial membrane potential was not changed in hoe-1(ΔNES) mutants. More characterization of mitochondrial function in hoe-1(ΔNES) mutants is required, such as OCR and mitochondrial morphology. It seems that hoe-1(ΔNES) mutants are smaller than wild-type animals.

    6. In fig 4a, Why the overall level of ATFS-1 is dramatically increased in hoe-1(ΔNES) mutants, this is not consistent with only two-fold up-regulation of atfs-1 transcript levels. The authors also would need to show the ATFS-1::GFP expression pattern in the nuo-6 mutants as a control.

  6. eLife

    Reviewer #3 (Public Review):

    Held and colleagues present numerous intriguing findings suggesting that the tRNA processing enzyme ELAC2/HOE-1 is required to activate the mtUPR in C. elegans. The hoe-1 gene encodes 2 proteins, one of which contains a mitochondrial targeting sequence (MTS) and a nuclear localization sequence (NLS). The other protein is similar but lacks the MTS. Thus, hoe-1 encodes proteins involved in tRNA processing in the nucleus and within mitochondria. It is intriguing that one or both of the proteins may be required for mtUPR activation. I have multiple concerns related to the experimental design and the interpretation. However, my major concern is that it remains unclear how HOE-1 regulates the mtUPR (DVE-1 or ATFS-1).

    Major concerns.

    -Figure 1. The authors use the transcriptional reporter hsp-6::gfp as a mtUPR reporter. However, in addition to requiring transcription for the hsp-6 promoter to induce the gfp mRNA, that mRNA must be synthesized. As HOE-1 is a tRNA processing enzyme likely required for protein synthesis, qRT-PCR analysis should be performed to quantify the effects of HOE-1 inhibition on the mtUPR transcription response. Thus the data supporting the claim that loss-of-function mutations in HOE-1 inhibit mtUPR-dependent transcription are weak and must be further substantiated.

    - Several groups have shown that inhibition of S6 kinase inhibits mtUPR activation. As HOE-1 is presumably required for protein synthesis, perhaps the mechanism is related? It would be good to know whether the inhibition of other genes affecting tRNA levels also impairs mtUPR or is specific to HOE-1.

    -It is my understanding that the HOE-1 protein with a mitochondrial targeting sequence is transcribed from the same gene as HOE-1 without the MTS. And, there are separate transcriptional start sites for each mRNA/protein. Considering the number of claims related to subcellular localization of HOE-1, the authors would need to determine if transcription from either site is altered during mitochondrial stress.

    - There is an over-reliance on the hoe-1(∆NES) strain which causes mtUPR activation. It remains unclear if nuclear accumulation is an event driving mtUPR activation or if the activation is simply an artifact of the ∆NES mutation. The hoe-1 loss of function studies are need to be further developed in order to interpret the hoe-1(∆NES) results. It remains possible that the ∆NES findings are simply an artifact of a neomorphic allele and do not inform on HOE-1 function.

    -The data suggesting that nuclear accumulation of HOE-1 is sufficient to activate mtUPR is relatively weak. Does HOE-1∆NES cause mitochondrial dysfunction which increases mtUPR activation? Potentially, HOE-1 lacking the nuclear export sequence may not accumulate within mitochondria and cause mitochondrial dysfunction. More in depth quantitative assessment of mitochondrial activity is required (TMRE images, oxygen consumption, etc). Alternatively, the ∆NES mutation could be combined with the ∆MTS mutation.

    -Fig 4. The authors generate a beautiful ATFS-1::mCherry fusion protein and demonstrate that accumulates within nuclei during mitochondrial stress. Does hoe-1 inhibition affect translation/synthesis of ATFS-1::mCherry or nuclear accumulation of ATFS-1::mCherry? Or, DVE-1?

    The mechanism by which hoe-1 impacts mtUPR is unclear.

  7. Version published to 10.1101/2021.06.22.449331v2 on bioRxiv
  8. Version published to 10.1101/2021.06.22.449331v1 on bioRxiv