The ribosomal RNA m 5 C methyltransferase NSUN-1 modulates healthspan and oogenesis in Caenorhabditis elegans

  1. Clemens Heissenberger
  2. Teresa L. Krammer
  3. Jarod A. Rollins
  4. Fabian Nagelreiter
  5. Isabella Stocker
  6. Ludivine Wacheul
  7. Anton Shpylovyi
  8. Santina Snow
  9. Johannes Grillari
  10. Aric N. Rogers
  11. Denis L.J. Lafontaine
  12. Markus Schosserer

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Abstract

Our knowledge about the repertoire of ribosomal RNA modifications and the enzymes responsible for installing them is constantly expanding. Previously, we reported that NSUN-5 is responsible for depositing m 5 C at position C2381 on the 26S rRNA in Caenorhabditis elegans .

Here, we show that NSUN-1 is writing the second known 26S rRNA m 5 C at position C2982. Depletion of nsun-1 or nsun-5 improved locomotion at midlife and resistance against heat stress, however, only soma-specific knockdown of nsun-1 extended lifespan. Moreover, soma-specific knockdown of nsun-1 reduced body size and impaired fecundity, suggesting non-cell-autonomous effects. While ribosome biogenesis and global protein synthesis were unaffected by nsun-1 depletion, translation of specific mRNAs was remodelled leading to reduced production of collagens, loss of structural integrity of the cuticle and impaired barrier function.

We conclude that loss of a single enzyme required for rRNA methylation has profound and highly specific effects on organismal physiology.

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  1. eLife

    ###This manuscript is in revision at eLife

    The decision letter after peer review, sent to the authors on April 8, 2020, follows.

    Summary

    Heissenberger et al. study how NSUN-1 impacts rRNA methylation and health in nematodes. Eukaryotic ribosomal RNAs undergo several modifications. Among these, there are two known m5C, located in highly conserved target sequences. Previous work from the authors characterised the mechanism underlying one of these modifications in worms (C2381), as well as its functional consequences on cellular and organismal homeostasis. The current work focuses on the second m5C, at position C2982, and identifies NSUN-1 as the putative rRNA methylase. This is a novel and potentially exciting finding.

    Using RNAi in two worm strains, the authors show that knocking down NSUN-1 expression, the specific C2982 m5C level is in part (not entirely) reduced. This assay proves sufficiency (but not necessity) of NSUN-1 to reduce m5C levels at C2982. While it is not clear why the authors do not use a complete knock out for NSUN-1 (is it lethal?), follow-up work using RNAi explores the phenotypic effects of lowered NSUN-1 levels.

    While somatic and germline reduction of m5C levels do not have an impact on worm lifespan, it does increase resistance to heat stress, slight increase in motor activity. Reducing NSUN-1 expression separately in germline and soma showed allegedly lifespan increase. Somatic reduction of NSUN-1 leads to changes in body size, oocyte maturation and fecundity, and has no effect on global protein translation. Analysis of polysome enrichment for specific mRNAs revealed that worms with low levels of NSUN-1 have altered translation of transcripts involved in cuticle collagen deposition.

    Major Points

    1. We are unconvinced by one of the major claims of this work, which is that C2982 has an impact on worm lifespan when expression is down in the soma. This claim does not seem to be strongly supported by the results shown. Were the replicates analysed separately or data from different assays pooled? Median lifespan appears the same between wt and RNAi worms. The survival raw data should be made available for reanalysis.
    2. It is not clear whether deletion mutants for NSUN-1 (e.g. nsun-1(tm6081)) are viable in C. elegans and if yes, what is their phenotype in the context of this study. If the deletion mutant is not available, can the authors generate a CRISPR line?
    3. Is there a relationship between the mRNAs selectively translated in the NSUN-1 RNAi treatment and in the NSUN-5 RNAi/mutant?
    4. The results shown in figure 1 draw a causal connection between NSUN-1 activity and C2982 based on exclusion: in other words, both NSUN-1 and NSUN-5 depletion lower the m5C peak by over 50%. Hence, since there are two m5C sites and one is written by NSUN-5, the other one must be written by NSUN-1. Is it possible that NSUN-1 may not be the only C2982 writer? Can the authors comment on this?
    5. Figure 4 analyzes the gonad and oocyte maturation. While the images are very convincing, it would be good to know how penetrant the phenotype is after analysis of a larger number of animals in each group.
    6. It is unclear how the observed translational remodeling that affects collagen deposition (demonstrated through the gonad extrusion and cuticle barrier phenotypes) is linked to oocyte maturation, or to heat stress resistance.
    7. The authors should indicate how many times the HPLC experiments were done.
    8. In Figure 3 the authors should indicate on each panel the age of the worms and at which stage the RNAi treatment was performed.
    9. The overall claim about behavior should be toned down as the RNAi line has no ovreall improvement, but only one time point shows a difference among the groups. From the text it is not clear what statistical test was used to analyze the differences in behavior among the groups.
    10. Although it may be hard to downregulate rRNAs by RNAi since they are so highly expressed, can the authors comment on whether 26S rRNA levels are reduced after RNAi and if yes to what degree?
    11. While the authors write that rrf-1 is required for amplification of the dsRNA signal specifically in the somatic tissues, this may not be completely accurate, as the Kumsta et al 2012 paper shows that rrf-1 affects both the soma and the germline. How does this affect the interpretation of the results?
    12. Is there a chance that 26S rRNA expression or differential methylation have a tissue-specific pattern (you use RT-qPCR from whole worms)?
    13. May NSUN-1 have pleiotropic effects independent of C2982 m5C?
  2. Version published to 10.1101/2020.03.16.993469 on bioRxiv