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 m5C at position C2381 on the 26S rRNA in Caenorhabditis elegans.
Here, we show that NSUN-1 is writing the second known 26S rRNA m5C 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.
Introduction
Ageing is a complex biological process, characterized by progressive aggravation of cellular homeostasis defects and accumulation of biomolecular damages. According to the ‘disposable soma theory’ of ageing, organisms may invest energy either in reproduction or in somatic maintenance (Kirkwood and Holliday, 1979). This explains why most lifespan-extending interventions come at the cost of decreased fecundity. De novo protein synthesis by ribosomes, the most energy-demanding process in living cells, affects the balance between ageing and reproduction. In fact, reduced overall protein synthesis was shown to extend lifespan in several model organisms, including the nematode Caenorhabditis elegans (Hansen et al., 2007; Pan et al., 2007; Syntichaki et al., 2007). Although some evidence indicates that a link between ribosome biogenesis and gonadogenesis in C. elegans may exist (Voutev et al., 2006), the precise relationship between these pathways in multicellular organisms is still poorly understood. It is conceivable that the optimal function of ribosomes, which requires the presence of ribosomal RNA (rRNA) and ribosomal protein (r-protein) modifications, is monitored by the cell at several stages during development. Thus, introduction of these modifications might participate in the control of cell fate and cell-cell interactions during development (Hokii et al., 2010; Voutev et al., 2006).
Eukaryotic ribosomes are composed of about 80 core r-proteins and four different rRNAs, which together are assembled into a highly sophisticated nanomachine carrying the essential functions of mRNA decoding, peptidyl transfer and peptidyl hydrolysis (Ban et al., 2014; Natchiar et al., 2017; Penzo et al., 2016; Sharma and Lafontaine, 2015; Sloan et al., 2017). Until recently, ribosomes were considered as static homogenous ribonucleoprotein complexes executing the translation of cellular information from mRNA to catalytically active or structural proteins. However, mounting evidence suggests the possibility of ribosomes being heterogeneous in composition with the possibility that some display differential translation with distinct affinity for particular mRNAs (Genuth and Barna, 2018). Such heterogeneity in composition may originate from the use of r-protein paralogs, r-protein post-translational modifications, or rRNA post-transcriptional modifications. Indeed, around 2% of all nucleotides of the four rRNAs are decorated with post-transcriptional modifications, which are introduced by specific enzymes such as dyskerin and fibrillarin, guided by specific small nucleolar RNAs (snoRNA) (Penzo et al., 2016; Sloan et al., 2017). The most abundant rRNA modifications are snoRNA-guided 2′O-methylations of nucleotide ribose moieties and isomerization of uridine to pseudouridine (Ψ). However, some base modifications, which occur less frequently than 2’O-methylations of ribose and pseudouridines, are installed by specific enzymes, which were largely assumed to be stand-alone rRNA methyltransferases. One exception is the acetyltransferase Kre33 (yeast)/NAT10 (human), which is guided by specialized box C/D snoRNPs (Sharma et al., 2015; Sleiman and Dragon, 2019). Most of these base modifications are introduced at sites close to the decoding site, the peptidyl transferase centre, or the subunit interface. Intriguingly, prokaryotes and eukaryotes share the majority of modifications located in the inner core of the ribosome (Natchiar et al., 2017).
In eukaryotes inspected so far, the large ribosomal subunit contains two m5C residues. This is notably the case in budding yeast (on 25S rRNA), in the nematode worm (26S) and in human cells (28S) (Sharma and Lafontaine, 2015). Rcm1/NSUN-5, an enzyme of the NOP2/Sun RNA methyltransferase family, is responsible for introducing m5C at residue C2278 and C2381 on 25S/26S rRNA in yeast and worms, respectively (Gigova et al., 2014; Schosserer et al., 2015; Sharma et al., 2013). Recently, our group and others identified the conserved target cytosines in humans and mice, namely C3782 and C3438, respectively (Janin et al. 2019, Heissenberger et al. 2019). We also reported that lack of this methylation is sufficient to alter ribosomal structure and ribosome fidelity during translation, while extending the lifespan and stress resistance of worms, flies and yeast (Schosserer et al., 2015). However, the identity of the second worm m5C rRNA methyltransferase remains unknown.
Here, we report that NSUN-1 is responsible for writing the second C. elegans 26S m5C (position C2982). We then investigate the physiological roles of NSUN-1, comparing them systematically to those of NSUN-5. We show that NSUN-1 and NSUN-5 distinctly modulate fundamental biological processes such as ageing, mobility, stress resistance and fecundity. In particular, depletion of nsun-1 impairs fecundity, gonad maturation and remodels translation of specific mRNAs leading to cuticle defects. We conclude that loss of NSUN-1 introducing a single rRNA modification is sufficient to profoundly and specifically alter ribosomal function and, consequently, essential cellular processes.
Results
NSUN-1 is responsible for writing m5C at position C2982 on C. elegans 26S rRNA
Previously, we showed that an m5C modification is introduced at position C2381 on the 26S rRNA of C. elegans large ribosomal subunit by NSUN-5 (Adamla et al. 2019; Schosserer et al. 2015), which is required to modulate animal lifespan and stress resistance (Schosserer et al., 2015). On this basis, we were interested to learn if other related rRNA methyltransferases in C. elegans might display similar properties.
Therefore, we investigated the RNA substrate of NSUN-1 (also formerly known as NOL-1, NOL-2 or W07E6.1) and its potential roles in worm physiology. NSUN-1 is also a member of the NOP2/Sun RNA methyltransferase family. Since there are only two known m5C residues on worm 26S rRNA (Sharma and Lafontaine, 2015; Trixl and Lusser, 2019), one of them at C2381, being installed by NSUN-5, we speculated that NSUN-1 might be required for introducing the second m5C residue at position C2982. Notably, both 26S m5C sites are localized close to important functional regions of the ribosome, and they are highly conserved during evolution (Fig. 1A,B).
In order to test if NSUN-1 is involved in large ribosomal subunit m5C methylation, 26S rRNA was purified from worms treated with siRNAs specific to NSUN-1-encoding mRNAs on sucrose gradients, digested to single nucleosides and analysed by quantitative HPLC. In our HPLC assay, the m5C nucleoside eluted at 12 min, as established with a m5C calibration control (data not shown).
The depletion of NSUN-1 was conducted in two genetic backgrounds: N2 (wildtype), and NL2099 (an RNAi-hypersensitive strain due to mutation in rrf-3) (Figure 1–figure supplement 1). Treating N2 worms with an empty control vector, not expressing any RNAi, did not significantly reduce the levels of 26S rRNA m5C methylation (Fig. 1C, 97% instead of 100%). Interestingly, treating N2 worms with an RNAi construct targeting nsun-1 led to a reduction of 26S rRNA m5C methylation by 35% (Fig. 1C). In the NL2099 strain, nsun-1 RNAi treatment also led to a reduction of 26S rRNA m5C methylation by 26% (Fig. 1D).
As there are only two known modified m5C residues on worm 26S rRNA, and since one of them is introduced by NSUN-5 (Adamla et al., 2019; Schosserer et al., 2015), a complete loss of NSUN-1 activity was expected to result in a 50% decrease in m5C methylation. However, protein depletion achieved with RNAi is usually not complete. It is not clear why the level of m5C depletion was not higher in the RNAi hypersensitive strain in comparison to the N2 strain; nonetheless, RNAi-mediated depletion of nsun-1 significantly reduced the levels of 26S rRNA m5C modification in both worm strains, thus, we conclude that NSUN-1 is responsible for 26S rRNA m5C methylation.
We analysed the 26S rRNA m5C levels in a nsun-5 deletion strain as control (strain JGG1, Fig 1E). In this case, we observed a near 2-fold reduction in methylation (58% residual), as expected from the known involvement of NSUN-5 in modification at position C2381. When nsun-1 was additionally depleted by RNAi in the nsun-5 knockout animals, the level of 26S rRNA m5C was further reduced to 43%, again in agreement with our conclusion that NSUN-1 is responsible for methylating the second position, C2982.
To further prove that NSUN-1 is not involved in C2381 modification, methylation levels at this position were specifically tested by Combined Bisulfite Restriction Analysis (COBRA) assay in animals depleted of nsun-1 or nsun-5. This method is based on bisulfite conversion of total RNA, followed by PCR amplification and restriction digest, yielding two bands in case of methylation at C2381 and three bands in case of non-methylation (Adamla et al., 2019). As expected, only nsun-5 depletion strongly reduced methylation at C2381, and there was no residual m5C2381 in the nsun-5 knockout strain, while nsun-1 RNAi had no effect on modification at this position (Fig. 1F). Bisulfite sequencing is well-known to be sensitive to RNA secondary structure (Warnecke et al., 2002), which likely explains why, despite repeated attempts, we could not monitor modification at position C2982 by use of this technique.
In conclusion, NSUN-1 and NSUN-5 are each responsible for installing one m5C onto the worm 26S rRNA, with NSUN-1 being responsible for position C2982 and NSUN-5 for position C2381 under the bona fide assumption that indeed only two m5C positions are present as described (Sharma and Lafontaine, 2015).
The somatic tissue-specific depletion of nsun-1 extends healthy lifespan
Next, we investigated if knockdown of nsun-1 modulates healthy lifespan in a similar fashion as nsun-5 (Schosserer et al., 2015). For this aim, we depleted nsun-1 by RNAi in N2 wildtype animals starting from day 0 of adulthood and, surprisingly, did not observe any extension of mean or maximum lifespan (Fig. 2A). Consequently, we also evaluated the stress resistance of adult worms upon nsun-1 or nsun-5 depletion, as an increased health at an advanced age often improves resilience to adverse events (Lithgow et al., 1994). Indeed, depletion of either nsun-1 or nsun-5 increased resistance to heat stress compared to the RNAi control (Fig. 2B). Similarly, we tracked the movement of animals exposed to either empty vector control or RNAi directed against nsun-1 or nsun-5 in a time course analysis, starting at day 1 of adulthood up to day 16. Interestingly, we observed a trend towards increased average speed in all three independent experiments at day 8 of adulthood in both nsun-1 (+47.8%) and nsun-5 (+34.7%) depleted animals compared to the control, and, to a lesser extent at day 12 (nsun-1 RNAi: +10.2%, nsun-5 RNAi: +73.5% compared to the control) (Fig. 2C). Thus, while nsun-1 knockdown does not extend lifespan, it improves the healthspan parameters thermotolerance and locomotion (Bansal et al., 2015; Rollins et al., 2017).
Intrigued that depletion of nsun-1 did not extend the lifespan of C. elegans in a similar fashion as nsun-5 did when whole adult animals were treated with RNAi, we reasoned that performing tissue-specific depletion of nsun-1 might help us to further elucidate a possible effect on lifespan. We focused on the comparison of the germline and somatic tissues, because somatic maintenance and ageing are evolutionarily tightly connected (Kirkwood and Holliday, 1979), and signals from the germline modulate C. elegans lifespan (Hsin and Kenyon, 1999). In addition, only loss of soma- but not germline-specific eIF4E isoforms, which are central regulators of cap-dependent translation, extend nematode lifespan (Syntichaki et al., 2007). To test if nsun-1 has similar specificity, we made use of worm strains sensitive to RNAi only in either the germline or somatic tissues. This is achieved, on the one hand by mutation of rrf-1, which is required for amplification of the dsRNA signal specifically in the somatic tissues (Kumsta and Hansen, 2012; Sijen et al., 2001), and, on the other hand by functional loss of the argonaute protein ppw-1 rendering the germline resistant to RNAi (Tijsterman et al., 2002). Interestingly, germline-specific nsun-1 RNAi had no effect on animal lifespan (Fig. 2D), but depletion of nsun-1 in somatic tissues reproducibly increased mean lifespan by ∼10% (Fig. 2E).
In conclusion, both NSUN-1 and NSUN-5 m5C rRNA methyltransferases modulate thermotolerance and mobility of wildtype nematodes at midlife. Whole-animal nsun-5 depletion expands mean lifespan by 17% (Schosserer et al., 2015), and in contrast to this, a 10% lifespan extension is only detected after depletion of nsun-1 specifically in the somatic tissues.
The somatic tissue-specific depletion of nsun-1 affects body size, fecundity, and gonad maturation
The ‘disposable soma theory’ of ageing generally posits that long-lived species exhibit impaired fecundity and reduced number of progeny. The underlying cause is that energy is invested in the maintenance of somatic tissues instead of rapid reproduction (Kirkwood and Holliday, 1979). In keeping with this theory, we expected that the absence of nsun-1 in somatic tissues, which increased longevity, may reduce fecundity. Therefore, we first measured the brood size upon nsun-1 and nsun-5 depletion by RNAi. After reaching adulthood but before egg-laying, worms were transferred to individual wells of cell culture plates containing NGM-agar and fed with bacteria expressing the specific RNAi or, as control, the empty vector. Egg production was impaired upon nsun-1 knockdown (reduced by 42%), but, surprisingly, this was not the case upon nsun-5 knockdown (reduced by 2%) (Fig. 3A). Egg production ceased rapidly after day one in all conditions (Figure 3–figure supplement 1A).
Thus far, all the experiments were performed on worms subjected to adult-onset nsun-1 knockdown, as animals depleted of nsun-1 during development were smaller and were infertile upon adulthood. To follow up on these observations, we measured mRNA expression levels of both m5C rRNA methyltransferases at different developmental stages including eggs, L1/L2 larvae, L3 larvae, L4 larvae and young adults. RT-qPCR indicated that both nsun-1 and nsun-5 mRNA levels constantly increase during development (Fig. 3B). The same observation applied to mRNA levels of nsun-2 and nsun-4 (Figure 3–figure supplement 1B), indicating that all four members of the NSUN-protein family might play important roles during development.
To further assess whether nsun-1 expression is indeed necessary for progressing faithfully through larval stages, we captured images of young adult animals subjected to larval-onset RNAi. The disparity in body size between RNAi control and nsun-1 RNAi was apparent, whereby nsun-1 depleted animals showed reduced length by approximately 20%. Interestingly, this reduced body size was not seen upon nsun-5 RNAi treatment (Fig. 3C,D).
In addition, we imaged 3-day old animals using differential interference contrast (DIC) microscopy. Worms subjected to nsun-1 knockdown displayed morphological alterations, specifically the gonad appeared severely distorted (Fig. 3E). In contrast, RNAi control and nsun-5 RNAi showed comparable morphology of distal and proximal gonads (Fig. 3E). Consequently, we hypothesized that nsun-1 RNAi treated worms might be arrested in early L4 larval stage when the gonad is not yet fully developed and animals still grow, instead of reaching normally adulthood after 3 days like RNAi control or nsun-5 RNAi treated nematodes. To test this, we used the TP12 kaIs12[col-19::GFP] translational reporter strain, which expresses COL-19::GFP specifically upon reaching adulthood, but not during larval stages. Surprisingly, larval-onset RNAi against nsun-1 or nsun-5 did not reveal differences in the expression of COL-19::GFP as compared to RNAi control, suggesting that neither nsun-1 nor nsun-5 induce larval arrest (Fig. 3F). Together with the reduced brood size upon adult-onset RNAi, these findings imply that loss of nsun-1 induces phenotypic changes in the reproductive organs of C. elegans independent of development.
Since knockdown of nsun-1 extended lifespan only when it was applied to somatic tissues, we hypothesized that body length might also be affected when these tissues are specifically targeted for depletion. To test this, we depleted nsun-1 specifically in the germline or in somatic tissues using tissue-specific RNAi strains and measured body size during three consecutive days after adulthood was reached. While germline-specific knockdown of nsun-1 did not induce any changes in body size (Fig. 3G), soma-specific knockdown revealed a decrease in body length by 23% on day 1, 11% on day 2 and 12% on day 3 compared to the RNAi control (Fig. 3H), phenocopying nsun-1 depletion in wildtype animals after whole animal RNAi. Similarly, no effect of nsun-1 knockdown was evident in another germline-specific RNAi strain, which was recently developed to enhance germline specificity (Zou et al., 2019) (Figure 3–figure supplement 1C).
In conclusion, nsun-1 but not nsun-5 depletion impairs body size and morphology of the gonad and leads to a significant reduction of brood size. Furthermore, these phenotypes are also observed when nsun-1 is specifically knocked-down in somatic tissues, but not when depleted in the germline only.
NSUN-1 is required for the transition of meiotic germ cells to mature oocytes
To further investigate the mechanisms underlying impaired fecundity upon nsun-1 knockdown, we analysed the morphology of the gonad in nsun-1 depleted animals in more detail. The germline of adult hermaphrodites resides within the two U-shaped arms of the gonad, which contains germ cells at various stages of differentiation (Fig. 4A). The gonad is sequentially developing from the proliferative germ cells near the distal tip cell, through the meiotic zone into the loop region, finally culminating in fully-formed oocytes in the proximal gonad (Pazdernik and Schedl, 2013). The limiting factor for fecundity in self-fertilizing hermaphrodites is sperm produced in the spermatheca (Hodgkin and Barnes, 1991).
Upon visualizing the germline cell nuclei with DAPI-staining, no oocytes were observed in worms after knockdown of nsun-1 in contrast to RNAi control treated animals (Fig. 4B). The mitotic zone at the distal end of the gonad appeared normal in nsun-1 depleted animals, whereas oocyte production starting at the pachytene zone was hampered. Analysis of GFP::RHO-1 and NMY-2:GFP expressing worm strains, which specifically express GFP in the germline, confirmed our observations (Fig. 4C, Figure 4–figure supplement 1). The gonads of control and nsun-5 RNAi treated animals appeared normal, clearly depicting the different stages of in-utero embryo development, whereas the germline of nsun-1 RNAi treated animals displayed a strikingly altered morphology.
Since other phenotypes observed upon nsun-1 depletion were detected in soma- but absent from germline-specific RNAi treated strains, we hypothesized that the somatic part of the gonad might specifically require NSUN-1 for normal oocyte production. Indeed, soma- specific depletion of nsun-1 phenocopied the distorted gonad morphology of wildtype animals exposed to nsun-1 RNAi (Fig. 4D). Remarkably, upon germline-specific knockdown of nsun-1, the gonad appeared completely unaffected (Fig. 4E; Figure 4–figure supplement 2).
NSUN-1 is not essential for pre-rRNA processing and global protein synthesis
Since the only known function of NSUN-1 and NSUN-5 is m5C methylation of rRNA, we reasoned that methylation-induced alterations of ribosome biogenesis and function might explain the observed phenotypes. Therefore, we tested if the presence of NSUN-1 or NSUN-5 is required for ribosomal subunit production and pre-rRNA processing. To this end, total RNA was extracted from worms treated with nsun-1 RNAi, separated by denaturing agarose gel electrophoresis and processed for northern blot analysis (Fig. 5A,B). Again, two reference worm strains were used (N2 and NL2099). Upon nsun-1 knockdown, we observed a mild accumulation of the primary pre-rRNA transcript and of its immediate derivative, collectively referred to as species “a” (Fig. 5A, (Bar et al., 2016; Saijou et al., 2004)), as well as a mild accumulation of the pre-rRNAs “b” and “c’” (Fig. 5A,B, see lane 1 and 3 as well as 5 and 6). Again, these findings were observed in both worm backgrounds tested, N2 and NL2099.
For comparison, we also analysed rRNA processing in nsun-5 deletion worms (JGG1 strain) in presence and absence of NSUN-1 (nsun-1 RNAi in JGG1). In both cases, we noted an important reduction in the overall production of ribosomal RNAs (Fig. 5B,C), with an apparent increase of rRNA degradation (seen as an increase in accumulation of metastable RNA fragments, in particular underneath the 18S rRNA). Furthermore, we observed that NSUN-1 is not required for mature rRNA production as shown by the unaffected levels of mature 18S and 26S rRNAs (Fig. 5C). This was confirmed by determining the 26S/18S ratio, which was 1.0 as expected since both rRNAs are produced from a single polycistronic transcript (Fig. 5C). The levels of the other two mature rRNAs (5S and 5.8S) were also unaffected (Figure 5–figure supplement 1). This behaviour was shown in both worm backgrounds, N2 and NL2099, used. The overall decrease in mature ribosome production observed in nsun-5 deletion worms did not affect the ratio of mature ribosomal subunits (26S/18S ratio of 1.0) (Fig. 5C). In agreement with the reduced amounts of 18S and 26S rRNA observed in nsun-5 deletion worms, total amounts of all precursors detected were reduced (Fig. 5B). Analysis of low molecular weight RNAs by acrylamide gel electrophoresis, revealed absence of NSUN-5 to severely inhibit processing in the internal transcribed spacer 2 (ITS2), which separates the 5.8S and 26S rRNAs on large precursors. This was illustrated by the accumulation of 3’-extended forms of 5.8S, and of short RNA degradation products (Figure 5–figure supplement 1, see lanes 3 and 4). Depletion of nsun-1 partially suppressed the effect of nsun-5 deletion: the overall production of mature rRNA and, in particular, the amount of mature 26S rRNA was increased (ratio of 1.2) (Fig. 5C). Consistently, the accumulation of 3’-extended forms of 5.8S and of short RNA degradation products was reduced (Figure 5–figure supplement 1).
In order to test if mature ribosomes of animals lacking any of the two m5C rRNA methyltransferases might be functionally defective, we analysed global protein synthesis by incorporation of puromycin in N2 worms treated with either RNAi control, nsun-1 or nsun-5 RNAi. Worms were exposed to puromycin for three hours at room temperature. Following lysis, puromycin incorporation was measured by western blot with an anti-puromycin antibody (Fig. 5D). Quantification of three independent experiments revealed no changes in global protein synthesis (Fig. 5E). We also performed polysome profiling which provides a “snapshot” of the pool of translationally active ribosomes. Comparison and quantification of profiles obtained from control and nsun-1 knockdown nematodes did not reveal any differences in the distribution of free subunits, monosomes and polysomes (Fig. 5F,G). This agrees with the absence of global protein translation inhibition in the metabolic (puromycin) labelling assay (Fig. 5E).
In conclusion, NSUN-1 is not required for rRNA processing nor for global translation. On the contrary, the amounts of ribosomal subunits were reduced in the absence of NSUN-5. The ribosomal biogenesis alterations observed upon nsun-5 depletion result from a combination of processing inhibitions in ITS2 and increased rRNA intermediates turnover. However, global translation was not detectably affected.
mRNAs encoding cuticle collagens are translationally repressed upon nsun-1knockdown
Since depletion of nsun-1 did not affect global protein synthesis, we hypothesized that loss of 26S rRNA m5C methylation might modulate the translation of specific mRNAs, as was previously observed after Rcm1 (NSUN-5 homolog) depletion in yeast (Schosserer et al., 2015). To test this possibility, we isolated mRNAs contained in the polysome fraction, systematically sequenced them by RNAseq and compared their respective abundance in polysomes versus total mRNAs contained in the lysate before fractionation. We considered only protein-coding mRNAs with a minimum fold change of 2 between translatome and transcriptome and an adjusted p-value cut-off at 0.05 (Fig. 6A; Supplemental Data File 1). The translation of more mRNAs was repressed (RNAi control: 599, nsun-1 RNAi: 536) than promoted (RNAi control: 94, nsun-1 RNAi: 84). Since the composition of 3’ UTRs can affect translation (Tushev et al., 2018), we analysed GC-content, length and minimal free folding energy of all coding, promoted and repressed mRNAs in our dataset (Fig. 6B). Interestingly, all three features significantly differed between RNAi control and nsun-1 RNAi in promoted and repressed mRNAs (p < 0.05), while they remained unchanged when analysing all coding mRNAs present in the dataset. These findings suggest that loss of nsun-1 causes the translation of specific subsets of mRNAs based on the composition and length of their 3’UTRs. Moreover, 3’UTRs of mRNAs repressed by nsun-1 depletion were exclusively and significantly enriched (p < 0.001) for several binding motifs of ASD-2, GLD-1 and RSP-3 (Supplemental Data File 2). All three RNA binding proteins play essential roles in C. elegans development (Lee and Schedl, 2010; Longman et al., 2000) and were not differentially regulated in the transcriptome between nol-1 RNAi and RNAi control (Supplementary Data File 3).
To further understand the mechanistic link between differential translation and the phenotypes observed upon nsun-1 knockdown, we performed GO-term enrichment analysis. Among others, GO terms associated with collagens, structural integrity of the cuticle and embryo development were significantly enriched amongst those mRNAs, which were translationally repressed by nsun-1 knockdown (Fig. 6C, Supplemental Data File 4).
Since three collagens (col-35, col-36 and col-37) were also among the five most strongly repressed genes upon loss of nsun-1 (Fig. 6A), we decided to assess whether collagen deposition is indeed altered. For this aim, we performed a specific histological staining protocol in which young collagen is strained blue and mature collagen is stained pink to brownish-red (Herovici, 1963; Teuscher et al., 2019). While young adult worms exposed to RNAi control showed presence of both young and mature collagen, animals subjected to nsun-1 RNAi displayed a strikingly reduced collagen deposition compared to the cytoplasmatic counter-stain (yellow) (Fig. 6D).
Interestingly, we repeatedly observed an increased fraction of animals displaying gonad extrusion upon nsun-1 RNAi, which might be caused by loss of cuticle structural integrity. To quantify this phenotype, we classified mid-aged animals according to the grade of gonad extrusion into three categories: i) no visible signs of gonad extrusion, ii) mild extrusion, or iii) severe extrusion (Figure 6–figure supplement 1A). Upon nsun-1 depletion, 134 of 220 animals (∼60%) showed mild to severe extrusion of the gonad (categories ii and iii), while no extrusion was observed in any of the tested 50 RNAi control nematodes (Fig. 6E). To assess further physiological consequences of altered collagen deposition, we tested cuticle barrier activity. This assay is based on the principle Hochst33342 dye being membrane permeable, but cuticle impermeable. As previously described by Ewald et al. 2015, worms were grouped into four categories: i) not permeable (no stained nuclei in the animal tail region), ii) mildly permeable (< 5 stained nuclei), iii) permeable (5-10 stained nuclei), or iv) highly permeable (>10 stained nuclei) (Figure 6–figure supplement 1B,C). Consistent with a change of several collagens, nsun-1 RNAi caused cuticle permeability (categories ii, iii and iv) in 26 of 46 animals (∼56%) compared to only 5 of 51 RNAi control animals (∼10%) (Fig. 6F).
Taken together, this indicates that NSUN-1 is partially required for translation of several cuticle collagens, which might not only explain the loss of gonad integrity, but also the increased cuticle permeability upon nsun-1 depletion. Moreover, several mRNAs whose translation depends on NSUN-1 are associated with embryogenesis and enriched for binding motifs of known regulators of nematode development.
Discussion
Although rRNAs are universally modified at functionally relevant positions, little is known about the biological functions and pathological roles of RNA methylation sites, or about their potential readers, writers and erasers (Sharma and Lafontaine, 2015). In this work we have investigated the molecular and physiological roles of two structurally related Sun-domain-containing RNA methyltransferases, NSUN-1 and NSUN-5 in C. elegans, each responsible for writing one m5C mark on 26S rRNA. We further describe NSUN-1 as a bona fide m5C rRNA writer enzyme that, if missing, directly entails physiological and developmental consequences. We conclude that, molecularly, loss of NSUN-1 function leads to translational remodelling with profound consequences on cell homeostasis, exemplified by loss of cuticle barrier function, and highly specific developmental defects, including oocyte maturation failure. We further suggest that extrusion of the gonad and loss of cuticle barrier function are directly caused by reduced expression of collagens, while the developmental defects are associated with altered expression of several important developmental regulators. We summarized the observed RNAi phenotypes in different strain backgrounds in Table 1.
According to the ‘disposable soma theory of aging’, a balance between somatic repair and reproduction exists. Depending on the current environment, an organism may direct the available energy either to maintenance of the germline and thereby ensuring efficient reproduction, or to the homeostasis of somatic cells including the prevention of DNA damage accumulation (Kirkwood and Holliday, 1979). While most of the known genetic and nutritional interventions increase the lifespan of organisms, they antagonistically also reduce growth, fecundity and body size (Kapahi, 2010; Kenyon et al., 1993). Indeed, reduction of overall protein synthesis by genetic, pharmacological or dietary interventions was reproducibly shown to extend longevity in different ageing model organisms (Chiocchetti et al., 2007; Curran and Ruvkun, 2007; Hansen et al., 2007; Kaeberlein et al., 2005; Masoro, 2005; Pan et al., 2007). These reports clearly established protein synthesis as an important regulator of the ageing process at the interface between somatic maintenance and reproduction. Thus, we were surprised to find that despite their ability to modulate healthy ageing and to methylate rRNA, neither NSUN-1 nor NSUN-5 were required for global protein synthesis in worms under the conditions tested. In the case of NSUN-5 depletion, we previously found overall translation to be decreased in mammalian cells (Heissenberger et al., 2019), but not in yeast (Schosserer et al., 2015). We reasoned that the higher complexity of mammalian ribosomes and associated factors might render them more vulnerable to alterations of rRNA secondary structure, for example by loss of a single base modification, than ribosomes from yeast and nematodes.
As ribosome biogenesis or global translational activity per se were not severely affected by loss of NSUN-1, the idea of a mechanism to ‘specialize’ the ribosome by rRNA modifications for selective mRNA translation seems attractive (Simsek and Barna, 2017). Indeed, lack of NSUN-1 and presumably the methylation at C2982 resulted in decreased translation of mRNAs containing GLD-1 and ASD-2 binding sites. These two proteins are closely related members of the STAR protein family involved in mRNA binding, splicing and nuclear export of mRNAs. While the molecular functions of ASD-2 are only poorly understood, the role of GLD-1 in embryonic development is well characterized (Lee and Schedl, 2010). GLD-1 levels are highest in the pachytene (also referred to as meiotic zone), where GLD-1 acts as a translational repressor of mRNAs modulating oogenesis. At the transition zone between the pachytene and the diplotene, GLD-1 levels sharply decrease and previously repressed mRNAs are consequently translated. Since the gonads of nsun-1 knockdown animals appeared defective precisely at this transition and GLD-1 target mRNAs were repressed, we speculate that either ribosomes lacking the methylation at C2982 have generally low affinity for these mRNAs, or that translational repression by GLD-1 is never fully relieved. Although the expression of GLD-1 itself was not differentially regulated between control and nsun-1 depleted animals at transcription or translation level (Supplemental Data File 1 and 3), multiple direct or indirect interactions with NSUN-1 to modulate ribosome function are still conceivable and will require further studies.
Previously, Curran and Ruvkun reported that depletion of nsun-1 (W07E6.1) by adult-onset RNAi was able to extend lifespan in C. elegans (Curran and Ruvkun, 2007). The authors used for their high throughput screen a strain carrying a mutation in the eri-1 gene, rendering it hypersensitive to RNAi in the whole body, but especially in neurons and in the somatic gonad (Kennedy et al., 2004). In this study, we conducted a whole-body knockdown in N2 wildtype animals, but could not verify these previous findings on lifespan, although thermotolerance and the health status of mid-aged nematodes, as assessed by quantifying locomotion behaviour, were elevated. However, when knocking-down nsun-1 specifically in somatic tissues, but not in the germline, we observed a lifespan extension. The N2 wildtype strain is usually resistant to RNAi in the somatic gonad and neurons. Thus, we hypothesize that depletion of nsun-1 specifically in the somatic part of the gonad is required for lifespan extension, which is only effectively realised in the eri-1 and ppw-1 mutant strains, but not in N2 wildtype animals. Intriguingly, these findings further suggest possible non-cell-autonomous effects of single RNA methylations, since modulation of NSUN-1 levels in somatic cells profoundly affected distinct cells of the germline.
The developing gonad of L1 larvae consists of two primordial germ cells and two surrounding somatic gonad precursor niche cells. The crosstalk between these two cell types, which form the germline and somatic part of the gonad at later stages of larval development, was already described to modulate ageing and stress responses. Laser depletion of both primordial germ cells extends lifespan via insulin/IGF-signalling, while animals with an additional depletion of the two somatic gonad precursor cells have a normal lifespan (Hsin and Kenyon, 1999). Of potential relevance to our study is a recent report by Ou and coworkers, who demonstrated that IFE-4 regulates the response to DNA damage in primordial germ cells in a non-cell-autonomous manner via FGF-like signalling. Soma- specific IFE-4 is involved in the specific translation of a subset of mRNA including egl-15. Thereby, IFE-4 regulates the activity of CEP-1/p53 in primordial germ cells despite not being present there (Ou et al., 2019). We thus hypothesize that selective translation of mRNAs by specialized ribosomes, either generated by association with translational regulators such as IFE-4, or by RNA modifications as described here, might serve as a general mechanism to tightly control essential cellular processes even in distinct cells and tissues.
Elucidating the precise mechanism is of importance, as human NSUN1 (also known as NOP2 or P120) was shown to be required for mammalian preimplantation development (Cui et al., 2016) and thus indicates evolutionary conservation. Cui and colleagues found that NSUN1 has an indispensable role during blastocyst development within their experimental setup. Additionally, other groups reported that low levels of NSUN1 reduce cell growth in leukaemia cells, which is in line with findings that NSUN1 promotes cell proliferation. Moreover, high NSUN1 expression results in increased tumour aggressiveness and augmented 5-azacytidine (5-AZA) resistance in two leukaemia cell lines (Bantis et al., 2004; Cheng et al., 2018; Saijo et al., 2001). Thus, NSUN1 might be considered as an example of ‘antagonistic pleiotropy’. According to this theory, genes can be indispensable early in life but negligible later, for instance after sexual reproduction. While NSUN1 appears to be essential for normal development, it might increase tumour aggressiveness later in life, especially in highly proliferative cells and tissues.
Material and Methods
Worm strains and culture conditions
The following C. elegans strains were used in this study: N2; JGG1 nsun-5(tm3898), SA115 unc-119(ed3), JJ1473 unc-119(ed3), TP12 kaIs12[col-19::GFP], DCL569 mkcSi13[Psun-1::rde-1::sun-1 3’UTR + unc-119(+)], NL2098 rrf-1(pk1417) and NL2550 ppw-1(pk2505). Worms were cultured following standard protocols on Escherichia coli OP50-seeded NGM agar plates at 20°C, unless indicated otherwise (Brenner, 1974).
RNAi knockdown
The nsun-1 RNAi clones was from the J. Ahringer library (Dong et al., 2003) and the nsun-5 RNAi clone from the M. Vidal library (Rual et al., 2004). For inactivating nsun-1 and nsun-5, feeding of double-stranded RNA expressed in bacteria was used (Timmons et al; 2001). Therefore, the HT115 strain of E. coli, carrying either the respective RNAi construct or the empty vector (L4440) as RNAi control, was cultured overnight in LB medium with ampicillin and tetracyclin at 37°C. Bacteria were harvested by centrifugation, resuspended in LB medium and either 100 µL (60 mm plates) or 400 µL (100 mm plates) were plated on NGM containing 1 mM isopropyl-b-D-thiogalactoside and 25 µg/mL carbenicillin. The plates were incubated at 37°C overnight and used within one week.
Larval-onset RNAi was achieved by bleaching adult animals. Released eggs were transferred directly to plates seeded with RNAi bacteria. Adulthood was usually reached after three days and animals were used for experiments when the RNAi control strain started to lay eggs.
In case of adult-onset RNAi, eggs were transferred to plates seeded with RNAi control bacteria. Animals were raised until egg production commenced and subsequently transferred to the respective RNAi bacteria.
Differential Interference Contrast (DIC) microscopy
Worms were paralyzed using 1 M sodium azide solution and mounted on 2% agar pads. Images were acquired on a Leica DMI6000B microscope with a 10x dry objective (NA 0.3) or a 63x glycerol objective (NA 1.3) in DIC brightfield mode. Cropping, insertion of scale bars and brightness and contrast adjustments were done with Image J (version 2.0.0-rc-65/1.51w; Java 1.8.0_162 [64-bit]).
Mobility
Animals were either synchronized by timed egg-lay (two replicates) or by hypochlorite treatment (one replicate) on RNAi control plates. When reaching adulthood, nematodes were transferred to RNAi plates. Every few days at regular intervals, plates were rocked in order to induce movement of animals and videos were subsequently recorded for one minute. Worms were transferred to fresh plates whenever necessary. At day 16 the vast majority of worms completely ceased movement, thus we did not include any later timepoints. Notably, we did not notice any obvious aversion behavior or elevated speed at young age upon nsun-1 or nsun-5 RNAi, which was previously shown to be present upon depletion of other components of the translational machinery (Melo and Ruvkun, 2012). Worm Lab version 4.1.1 was used to track individual animals and calculate the average speed.
Lifespan assays
Lifespan measurement was conducted as previously described (Schosserer et al., 2015). For lifespan assays, 90 adults per condition were transferred to plates seeded with the respective RNAi bacteria (control, nsun-1, nsun-5). Wildtype worms were pre-synchronised on NGM plates seeded with UV-killed OP50 bacteria. 50 adult worms were transferred to NGM plates and allowed to lay eggs for 15 h; then the adult worms were removed. Synchronisation by timed egg lay was performed 72 h after the pre-synchronisation by transferring 350 gravid worms from the pre-synchronisation to fresh NGM plates seeded with RNAi control bacteriaand allowed to lay eggs for four hours. After 68 h, 90 young adult worms per condition were placed on fresh NGM plates containing 5 mL NGM, 100 µL bacterial suspension and 50 μg FUDR. This day represents day 0 in the lifespan measurement. Worms were scored as “censored” or “dead” every two to four days. Nematodes were scored as “censored” if they had crawled off the plate, were missing or died due to other causes than ageing, such as gonad extrusion. Animals were transferred to fresh plates every 3–7 days depending on the availability of the bacterial food source. Lifespans were performed at 20°C. Kaplan-Meier survival curves were plotted and log-rank statistics were calculated.
Thermotolerance
Thermotolerance was assessed as previously described (Vieira et al., 2017). Animals were synchronized by hypochlorite treatment and released eggs were transferred to NGM plates seeded with RNAi control bacteria and kept at 20°C. After 48 h, L4 animals were picked on RNAi control, nsun-1 or nsun-5 RNAi plates and exposed to RNAi for approximately three days (68 hours). Subsequently, plates were transferred to 35°C and scored every 1-2 h for survival.
Body size
Worms were synchronized by hypochlorite treatment and incubated in liquid S-Basal medium overnight. On the following day, eggs/L1 were transferred to RNAi plates (RNAi control, nsun-1 and nsun-5 RNAi). Three days later, worms were transferred to agar pads and paralyzed using sodium azide and visualized using DIC microscopy (see above).
Brood size analysis
Worms were synchronised by treatment with hypochlorite solution and incubated in S-Basal at room temperature overnight. L1 larvae were subsequently transferred to NGM plates seeded with RNAi control bacteria. After 48 h, L4 animals were transferred to individual wells of a 24-well plate seeded with the respective RNAi bacteria (HT115, nsun-1, nsun-5). Each well contained 1.5 mL of NGM agar and 3 μL of bacterial suspension (1:2 dilution in S-Basal). Worms were transferred to a new well every day for four consecutive days and total progeny of individual animals was counted. Per condition and experiment, five worms were analysed.
Global protein synthesis by puromycin incorporation
Puromycin incorporation was measured as previously described (Tiku et al., 2018) with minor modifications. Heat-inactivated OP-50 (75°C, 40 min) were provided as food source during pulse labelling. As negative controls, RNAi control treated worms were used either without addition of puromycin or by pulse-labelling at 4°C instead of 20°C. Around 100 animals per condition were harvested for western blot analysis. Lysis was done directly in SDS loading dye (60 µM Tris/HCl pH 6.8, 2% SDS, 10% glycerol, 0.0125% bromophenol blue and 1.25% β-mercaptoethanol). Worms in SDS loading dye were homogenized with a pellet pestle for 1 min. Then, the samples were heated to 95°C and loaded on 4-15% Mini-PROTEAN® TGX gels (BioRad) in Laemmli-Buffer (25 mM Tris, 250 mM glycine and 0.1% SDS). Protein bands were transferred to PVDF-membranes (Bio Rad) at 25 V and 1.3 A for 3 min. After blocking with 3% milk in PBS, the membrane was incubated overnight at 4°C with a mixture of anti-Histone H3 (Abcam ab1791, 1:4000) and anti-puromycin (Millipore 12D10, 1:10000). After washing and secondary antibody incubation (IRDye680RD and IRDye800CW, 1:10000), the membrane was scanned on the Odyssey Infrared Imager (LI-COR). Quantification of band intensities was performed in Image J (version 2.0.0-rc- 65/1.51w; Java 1.8.0_162 [64-bit]).
Polysome profling
Two-day-old adult worms were used to generate polysome profiles as previously described (Rogers et al., 2011). One hundred microliter worm-pellet were homogenized on ice in 300 µL of solubilisation buffer (300 mM NaCl, 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 1 mM EGTA, 200 µg/ml heparin, 400 U/ml RNAsin, 1.0 mM phenylmethylsulfonyl fluoride, 0.2 mg/ml cycloheximide, 1% Triton X-100, 0.1% sodium deoxycholate) using a pellet pestle. 700 µl additional solubilisation buffer were added, vortexed briefly, and placed on ice for 10 min before centrifugation at 20.000 g for 15 min at 4°C. Approximately 0.9 mL of the supernatant was applied to the top of a linear 10-50% sucrose gradient in high salt resolving buffer (140 mM NaCl, 25 mM Tris-HCl (pH 8.0), 10 mM MgCl2) and centrifuged in a Beckman SW41Ti rotor (Beckman Coulter, Fullerton, CA, USA) at 180.000 g for 90 min at 4°C.
RNA Seq
Gradients were fractionated while continuously monitoring the absorbance at 260 nm. Trizol LS (Life Technologies) was immediately added to collected fractions and RNA was isolated following the manufacturer’s protocol. PolyA-selection, generation of a strand-specific cDNA library and sequencing on the HiSeq 4000 platform (Illumina) using the 50 bp SR mode was performed by GATC Biotech (Konstanz, Germany). At least 30 million reads were generated per sample.
FASTQ Trimmer by column (Galaxy Version 1.0.0) was used to remove the first 12 bases from the 5’ end of each read due to an obvious base bias in this region, as detected by FastQC (Galaxy Version 0.69). Filter by quality (Galaxy Version 1.0.0) was performed using a cut-off value of 20 and only reads with a maximum number of 8 bases with quality lower than the cut-off value were retained. RNA STAR (Galaxy Version 2.6.0b-1) was used to align reads to the WBcel235 reference genome using the default options. Aligned reads with a minimum alignment quality of 10 were counted using htseq-count (Galaxy Version 0.9.1).
Differential expression was analyzed using the DEseq2 package in R. The contrast (batch = biological replicate, condition = sample description) was applied to compare the polysome fraction to the total RNA of either RNAi control or nsun-1 RNAi treated samples. Afterwards, results were filtered in R to contain only protein-coding genes (according to ENSEMBL annotation), genes with detectable expression (base mean > 1), a fold change of > 2 (log2FC > 1) and an adjusted p-value of < 0.05. Vulcano plots were generated in R using the EnhancedVulcano package, labelling the top 5 up- and down-regulated genes respectively.
GO term enrichment using DAVID (version 6.7) was performed as previously described (Rollins et al., 2019). Only protein-coding genes with detectable expression (base mean > 1), a fold change of > 2 and an adjusted p-value of < 0.10 were considered. For visualization, only the broadest GO terms of the GOTERM_BP_FAT, GOTERM_MF_FAT and GOTERM_CC_FAT categories, which were still significantly enriched (FDR < 0.05), are shown while similar terms based on the same subset of genes but lower in hierarchy were manually removed. Full results are contained in the supplements. UTR characterization and RBP motif enrichment were performed as previously described (Rollins et al., 2019) using only protein-coding genes with detectable expression (base mean > 1), a fold change of > 2 and an adjusted p-value of < 0.10.
The raw and processed sequencing data are available from the Gene Expression Omnibus database (https://www.ncbi.nlm.nih.gov/geo) under accession GSE143618.
The R-script for analyzing RNA-seq data is provided as Supplemental Data File 5.
RT-qPCR
Samples were collected by either transferring worms individually into 1.5 mL tubes or by washing them off NGM plates using S-Basal. After three washing steps with S-Basal, 300 μL TRIzol® LS Reagent were added to approximately 100 µL residual S-Basal including worms. Subsequently, worms were homogenised with a pellet pestle for one minute, 600 μl TRIzol® LS Reagent were added and the sample was vortexed for five minutes at room temperature. Total RNA was isolated using Direct-zol™ RNA MiniPrep Kit (Zymo) according to the instructions by the manufacturer. For cDNA synthesis, 500 ng RNA were converted into cDNA using the Applied Biosystems™ High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). cDNA was amplified from total RNA using random primers. RT-qPCR was performed on a Rotor-Gene Q (QIAGEN) using HOT FIREPol® EvaGreen® qPCR Mix. The absolute amounts of mRNAs were calculated by computing a standard curve and the resulting copy numbers were normalized to the housekeeping genes act-1 and tba-1. The following primers were used: nsun-1: 5’-TCGCCGAGATCCACAGAAAT-3’ (sense) and 5’-CCACGTTCATTCCACGGTTG-3’ (antisense); nsun-2: 5’-GCTTAAACGAGAGACGGGAGTT-3’ (sense) and 5’-CACCAGTATCCTGGGCGTG-3’ (antisense); nsun-4: 5’-TGTTGGATATGTGTGCGGCT-3’ (sense) and 5’-GCGTCCTTGCGTTTTAGGAC-3’ (antisense); nsun-5: 5’-GGCCAAGGAGAAAAGTGTG-3’ (sense) and 5’-GATCCACCGATATTCGCAT-3’ (antisense); act-1: 5’-CTACGAACTTCCTGACGGACAAG-3’ (sense) and 5’-CCGGCGGACTCCATACC-3’ (antisense) and tba-1: 5’-TCAACACTGCCATCGCCGCC-3’ (sense) and 5’-TCCAAGCGAGACCAGGCTTCAG-3’ (antisense).
For measuring mRNA expression during development, worms were synchronised by treatment with hypochlorite solution and the released eggs were subsequently transferred to four separate NGM plates seeded with UV-killed OP50 bacteria. Samples were taken from eggs immediately after bleaching, L1/L2 (20 h after bleaching), L3 (32 h after bleaching), L4 (46 h after bleaching) and young adults (60 h after bleaching).
3-D ribosome structure
The PyMOL Molecular Graphics System (Version 2.0) was used. The structure was modelled on the human 80S ribosome (PDB 6EK0).
m5C detection by COBRA assay
NSUN-5 activity was measured by the COBRA assay as previously described (Adamla et al., 2019) using the following primers: 5’-GGGAGTAATTATGATTTTTCTAAGGTAG-3’ (sense) and 5’-ATAATAAATAAAAACAATAAAAATCTCACTAATCCATTCATACAC-3’ (antisense).
HPLC analysis of m5C
13-15µg 26S purified on sucrose gradient were digested to nucleosides and analyzed by HPLC. Peaks elutes at 12 min and as a control, a commercial 5-methylcytidine (NM03720, CarboSynth) was used. For quantification of m5C peak area, the peak was normalized to either the peak eluting at 16 min (asterisk on the Figure), or to the peak eluting at 8 min (U), with similar results. The results are shown for normalization to the peak eluting at 16 min.
Pre-rRNA processing analysis
For analysis of high–molecular weight RNA species, 3 µg total RNA was resolved on a denaturing agarose gel (6% formaldehyde/1.2% agarose) and migrated for 16 h at 65 volts. Agarose gels were transferred by capillarity onto Hybond-N+ membranes. The membrane was prehybridized for 1 h at 65°C in 50% formamide, 5x SSPE, 5x Denhardt’s solution, 1% SDS (w/v) and 200 µg/ml fish sperm DNA solution (Roche). The 32P-labeled oligonucleotide probe (LD2648 (ITS1): CACTCAACTGACCGTGAAGCCAGTCG; LD2649 (ITS2): GGACAAGATCAGTATGCCGAGACGCG) was added and incubated for 1 h at 65°C and then overnight at 37°C. For analysis of low molecular weight RNA species, northern blots were exposed to Fuji imaging plates (Fujifilm) and signals acquired with a Phosphorimager (FLA-7000; Fujifilm).
Statistics and sample size estimation
No explicit power analysis was used. Sample sizes estimations were partially based on our own previous empirical experience with the respective assays, as well as the cited literature.
No systematic blinding of group allocation was used, but samples were always analysed in a random order. Nematodes were randomly assigned to the experimental groups. All lifespan, stress resistance and locomotion experiments were performed by at least two different operators.
Most experiments were performed in three independent experiments, unless stated otherwise in the figure legend. Independent experiments were always initiated at different days and thus always resemble different batches of nematodes. Some experiments (RNA isolation for RNA-seq, HPLC analysis of m5C, pre-rRNA processing analysis) were performed once with all frozen independent batches of nematodes to minimize technical variation. No outliers were detected or removed. Criteria for censoring animals for lifespan, stress resistance and locomotion experiments are indicated in the respective chapters.
Statistical tests used, exact values of N, definitions of center, methods of multiple test correction, and dispersion and precision measures are indicated in the respective figure legends. P-value thresholds were defined as *P < 0.05, **P < 0.01 and ***P < 0.001. For RNA-seq, statistical tests and p-value thresholds are explained in detail in the “RNA-seq” chapter.
Author Contributions
Planned experiments: C.H., T.L.K., J.A.R., J.G., A.N.R., D.L.J.L., M.S., performed experiments: C.H., T.L.K., I.S., A.S., S.S., M.S., analysed data: C.H., T.L.K., J.A.R., L.W., D.L.J.L., M.S., wrote manuscript: C.H., D.L.J.L., M.S., supervised the project: M.S.
Figure Supplements
Source Data Files for Figures
Figure 2–Source Data 1: Summary of individual lifespan and thermotolerance experiments
Acknowledgements
We are grateful to Tamás Barnabás Könye for technical assistance; the BOKU-VIBT Imaging Center for technical support with microscopy. This work was supported by the Austrian Science Fund (FWF) and Herzfelder’sche Familienstiftung [P30623 to M.S.], Hochschuljubiläumsstiftung der Stadt Wien [H-327123/2018 to M.S.], and the Austrian Science Fund (FWF) [I2514 to J.G.]. Research reported in this publication was supported by the James L. Boyer Fellowship at the MDI Biological Laboratory to M.S. Research conducted in the labs of A.N.R and J.A.R. was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant numbers P20GM103423 and P20GM104318. Research in the Lab of D.L.J.L. is supported by the Belgian Fonds de la Recherche Scientifique (F.R.S./FNRS), the Université Libre de Bruxelles (ULB), the Région Wallonne (DGO6) [grant RIBOcancer n°1810070], the Fonds Jean Brachet, the International Brachet Stiftung, and the Epitran COST action (CA16120). F.N. is a fellow of the international PhD programme “BioToP-Biomolecular Technology of Proteins”, funded by the Austrian Science Fund (FWF) [W1224 to J.G.]. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).