Box C/D Small Nucleolar Ribonucleoproteins Regulate Mitochondrial Surveillance and Innate Immunity

  1. Elissa Tjahjono
  2. Alexey V. Revtovich
  3. Natalia V. Kirienko

  • Curated by eLife

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    Evaluation Summary:

    This manuscript addresses a potential role for the box C/D Small Nucleolar Ribonucleoprotein complex at the intersection of mitochondrial homeostasis and cellular responses to infection and environmental stress in the context of the C. elegans system. The presented data can be explained in multiple ways and can serve as starting point for further research in this interesting and important area.

    “(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

Monitoring of mitochondrial functions is crucial for organismal survival. This task is performed by mitochondrial surveillance or quality control pathways, which are activated by signals originating from mitochondria and relayed to the nucleus (retrograde response) to start the transcription of protective genes. In Caenorhabditis elegans , several systems exist, including the UPR mt , MAPK mt , and the ESRE pathway. These pathways are highly conserved and their loss results in compromised survival following mitochondrial stress.

In this study, we found a novel interaction between the box C/D snoRNA core proteins (snoRNPs) and mitochondrial surveillance and innate immunity pathways. We showed that C/D snoRNPs are required for the full expressions of UPR mt and ESRE upon stress. Meanwhile, we found that the loss of C/D snoRNPs increased immune responses. Understanding the “molecular switch” mechanisms of interplay between these pathways may be important for understanding of multifactorial processes, including response to infection or aging.

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  1. eLife

    Evaluation Summary:

    This manuscript addresses a potential role for the box C/D Small Nucleolar Ribonucleoprotein complex at the intersection of mitochondrial homeostasis and cellular responses to infection and environmental stress in the context of the C. elegans system. The presented data can be explained in multiple ways and can serve as starting point for further research in this interesting and important area.

    “(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.)

  2. eLife

    Reviewer #1 (Public Review):

    In the presented manuscript entitled Box C/D Small Nucleolar 1 Ribonucleoproteins
    2 Regulate Mitochondrial Surveillance and Innate Immunity by Tjahjono et al., the authors describe data supporting novel interactions between the box C/D snoRNA core proteins (snoRNPs), mitochondrial surveillance processes and innate immunity in C. elegans. They find that C/D snoRNPs contribute to the regulation of the unfolded protein response in mitochondria (UPRmt) and the ethanol stress response (ESRE). Loss of C/D snoRNPs is further shown to increase expression of the innate immune reporters irg-5::gfp and irg-1::gfp. The authors conclude that Box C/d snoRNPs are upstream regulators of both mitochondrial surveillance and innate immunity, and as such potentially important to combat aging-associated and infectious diseases.

    The conclusions presented in this paper are not always supported by the presented data. Reasonable alternative explanations for the observed results are not consequently considered. Further, some key controls are missing, which complicates and inclusive full assessment of the data quality and conclusions, and reduces the anticipated impact on the field significantly.

    This study and all most conclusions rely on experiments that used the technique of RNAi feeding, which is well established in C. elegans. While the authors highlight in the materials and methods section that each RNAi was sequence-verified, on-target effects of individual RNAis in the used conditions are not assessed. qPCR-based verification of key RNAis in both L1 and L3 animals would need to be performed to better understand the role of the box C/D snoRNPs in the tested assays.

    The authors used RNAi targeting ruvb-1 to demonstrate to assess of nucleolar localization of snoRNPs is required to trigger the studied phenotype. However, the authors didn't show that the ruvb-1 RNAi indeed knocks down this gene, nor do they consider ruvb-1-independent nucleolar localization of snoRNPs. To support their conclusions, the authors would need to perform subcellular fractionation / western blot or immunofluorescence staining to provide further evidence when and where the investigated snoRNPs are required.

    The authors did not validate/test that the cycloheximide treatment results in the intended inhibition of translation. Data shown e.g. in Fig. S3 or Fig. 7A, showing an increase in Pirg-1::gfp-dependent GFP expression, indicates that cycloheximide is not interfering with translational processes as expected. The utilized reporter strains, although presented as transcriptional reporters, require the transcription and translation of a gfp gene in order to give a positive result in the reporter assays. Thus, less GFP signal may always arise as a consequence of general translation inhibition. In this reviewer's opinion, this aspect is not sufficiently considered.

    Conceptually, this reviewer feels that it is a stretch to conclude from assessing a single reporter per cellular process that Box C/D snoRNPs are master upstream regulators of mitochondria and innate immune mechanisms.

  3. eLife

    Reviewer #2 (Public Review):

    Tjahjono et al. identify a possible role for Box C/D snoRNP proteins in regulating a transcriptional response to reactive oxygen species the authors have previously described as being mediated by the ESRE network. In contrast to other known canonical mitochondrial stress pathways discussed by the authors-in particular, UPRmt and mitophagy-the possible role of the ESRE network response in mitochondrial homeostasis remains less established, with prior studies also conducted in this area also conducted by the authors. Thus, the current study aims to better understand how transcription of genes with an ESRE motif might be coupled to cellular stress, the authors began with a biochemical strategy to isolate C. elegans proteins that directly bind to the ESRE motif under conditions of stress that activate the ESRE-regulated genes. The authors report 75 candidate interacting proteins were isolated. The authors then shift to functional validation of these candidates using a combination of RNAi knockdown of corresponding candidate genes in C. elegans and a readout of GFP reporter genes corresponding to ESRE-dependent transcription and other stress response pathways. There is no further characterization or validation of the biochemical interactions in an in vitro or cellular context.

    The authors identify fib-1 and nol-56, two members of the box C/D snoRNP complex, as genes that when subjected to RNAi, result in diminished expression of an ESRE reporter gene in response to rotenone. nol-58 is then also tested by RNAi and found to similarly cause diminished expression of the reporter gene. The authors then conclude: "Since knockdown of any of the genes for these three proteins reduces ESRE signaling, it seems likely that the snoRNAP complex as a whole is binding to ESRE." The three genes encoding protein components of the box C/D snoRNP complex are known to be essential, thus complicating the interpretation of RNAi knockdown, which the authors note caused reduced growth and development of larval animals and thus alter the stage at which RNAi is initiated, observing similar effects on ESRE reporter gene expression when RNAi is initiated at a later, L3 larval stage. The authors then proceed to perform the same RNAi analysis using the Phsp-6::GFP (UPRmt reporter) and observed reduced GFP expression in response to UPRmt induction. An additional set of experiments examine Ptbb-6::GFP reporter gene expression, which has been recently described as being regulated by a PMK-3-dependent mitochondrial homeostasis pathway. The authors further performed RNAi of ruvb-1, which promotes box C/D snoRNAPs assembly and localization to nucleoli and observe that this also reduced ESRE-dependent GFP reporter gene expression though induction of the UPRmt was not affected. The authors also investigated RNAi of box H/ACA snoRNPs and did not observe effect, leading them to conclude that the "function of box C/D snoRNAPs in regulating mitochondrial homeostasis is specific." The authors also examine the effect of RNAi on genes involved in translation initiation and observed mixed effects and then treated animals with cycloheximide and did not observe changes. The authors then examine how previously characterized immunity reporter gene expression is affected by RNAi of box C/D snoRNP protein-encoding genes, with select genes exhibiting increased expression under these conditions. A single set of experiments outside of GFP reporter gene expression are conducted to evaluate how RNAi of box C/D snoRNP protein-encoding genes affects C. elegans physiology. Susceptibility to killing in liquid, conditions previously found to involve ESRE network gene expression, was compromised by RNAi of box C/D snoRNP protein-encoding genes, whereas susceptibility to Pseudomonas aeruginosa infection appeared to decrease with the same treatment.

    The authors have addressed an interesting area of cell biology in the context of the C. elegans system. The data provided are highly preliminary and lead to some interesting hypotheses, but the data are not sufficient to support the conclusions, and in general, there is a lack of rigor and overreliance on assumptions about the experimental methods being employed that require further validation.

    Specific conceptual and experimental concerns:

    1. The manuscript begins with biochemical characterization of the interaction of FIB-1 and NOL-56 with the ESRE motif. Are we to conclude that this was a non-specific interaction or a physiologically relevant interaction? Is there further validation in vitro or in vivo? Is the conclusion that FIB-1, NOL-56, and NOL-58 function in a snoRNP complex or is this a non-canonical activity? Do these proteins then somehow interact with the ESRE motif?

    2. RNAi of GFP reporter transgenes are subject to effects such as transgene silencing and other artifacts and should be corroborated by other methods such as qPCR. Moreover, while reporter genes are useful tools, it would seem that some transcriptome-wide-based comparisons should be also conducted to test and/or confirm hypotheses about whether knockdown of specific genes affects certain pathways. Generally, some sort of additional correlation that a pathway is being activated, e.g. change in localization of ATFS-1 in the UPRmt, greatly enhances the sense that a stress pathway is being activated.

    3. A critical issue that is only addressed obliquely by RNAi starting and L1 and L3 larval stages is that the genes studied, fib-1, nol-56, nol-58, are essential and RNAi of these have multiple pleiotropies. This seriously limits the interpretations that can be made of effects on gene expression, as animals are not developing and growing normally with essential gene inactivation having unpredictable effects. More importantly, the functional validation in liquid killing is particularly difficult to interpret as the effects of RNAi knockdown of essential genes may simply add sickness to the toxic effects of liquid killing. In turn, extended survival in the other Pseudomonas assay may reflect survival advantage of growing more slowly in those assays. Thus, what is lacking is a clear physiological assay or functional readout to corroborate the GFP reporter gene effects. One might expect altered physiological responses to mitochondrial toxins, but again, interpretation will be clouded by the essential nature of these genes.

    In summary, the manuscript presents some intriguing observations in an important area of biology intersecting at mitochondrial homeostasis and the response to infection and environmental stress, but it is difficult to know what one can conclude without invoking a number of unjustified assumptions from these preliminary observations.

  4. Version published to 10.1101/2021.04.28.441759 on bioRxiv