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Forum du site CaptaiNaruto. A photographic journey through the late visionary designer's fashion shows from to offers insight into his provocative couture style and performance venues, in a visual tribute based on input by his closest collaborators, By applying the same method to C. Thus, not only the modifying enzyme, but also the target site and probably molecular mechanism are highly conserved between these two organisms.

To demonstrate that methylation of C and no other molecular activity of Rcm1 is required for Rcm1-mediated lifespan extension, we performed chronological lifespan assays with yeast strains in which all rDNA repeats have been deleted. These data suggest that the lack of NSUN5 methylation activity towards a single nucleotide of rRNA is indeed sufficient for the observed lifespan modulation. A recent report suggests that the loss of a cluster of methylations including C leads to structural changes and ribosome instability under high-salt conditions To investigate if there are any conformational changes of the ribosome as a consequence of the loss of Rcm1 function alone, we performed structural probing employing the single-strand specific chemical probe dimethyl sulfate DMS.

Ribosomes isolated from yeast wt or from rcm1 -knockout cells were analysed in the presence or absence of oxidative stress Fig. Thus, the loss of Rcm1-mediated methylation of C leads to a transient structural change in the presence of H 2 O 2 , likely leading to a more relaxed 25S rRNA fold in the vicinity of C A and C denote dideoxy-sequencing lanes of untreated 25S rRNA and serve as marker for identifying DMS-reactive sites at the nucleotide resolution level.

The positions of some DMS-reactive adenosines are indicated on the right. DMS-reactive sites that change their reactivity only in the knockout strain during stress in a time-dependent manner are indicated by solid arrows. Additional positions that can be modified by DMS but do not show a differential pattern are indicated by open arrows. The primer extension stop intensities of DMS-reactive nucleobases in the absence of H 2 O 2 was taken as 1.

Values shown represent the mean and the s. The three-dimensional architecture was generated from pdb file 35UI. We next investigated which functional changes occur in translating ribosomes as a consequence of the Rcm1-mediated structural alterations of rRNA. Therefore, we performed polysome profile analysis, which provides a snapshot of the translationally active ribosome population Interestingly, comparison of the profiles obtained from wt and rcm1 -knockout cells did not show differences in the distribution of polysomes, 80S monosomes and free subunits, as previously reported 28 Fig.

Ribosomal profiles provide a snapshot of the translational status and are obtained by UVtracing of polysomes, single 80S ribosomes and free LSU and SSU populations, respectively, separated on a continuous sucrose gradient. In this case, Rcm1 deletion and oxidative stress decrease normalized reporter luminescence. However, as rcm1 -dependent conformational changes of the ribosome occur only on oxidative stress, we tested if also the polysome profiles were influenced by ROS using a single-dose treatment of 0.

This suggests that the presence of C methylation in wt cells limits, while loss of C methylation in rcm1 -knockout cells intensifies the shift from polysomes towards more 80S monosomes on acute stress. To investigate the functional consequences that may result from lack of C methylation, we tested translational fidelity of ribosomes lacking C methylations using a luciferase reporter construct with a premature termination codon PTC.

Rcm1 knockout promoted read-through of the premature stop codon by a factor of 2, compared with wt cells. This resembled increased read-through of the PTC reporter induced by oxidative stress in wt yeast Fig.

Since it has been previously reported that oxidative stress alters the fidelity of mRNA translation, increasing misreading and stop codon read-through 13 , 16 , these results suggest that the absence of C methylation resembles an activated cell status that counteracts oxidative stress.

To test this idea, we analysed the mRNA recruitment pattern into polysomes versus total cellular mRNAs under different oxidative stress conditions by microarrays. Expressed total mRNAs transcriptome and actively translated mRNAs within polysomes translatome were isolated from whole yeast cell lysates and analysed on microarrays in biological duplicates. TE is the ratio of the expression of a specific gene in the translatome versus the transcriptome.

In this scenario, the reduced availability of Rcm1 causes reduction of C methylation and structural alterations in the adjacent rRNA region. This alters the translational fidelity accompanied by mRNA recruitment in a similar fashion to oxidative stress, leading to increased stress resistance and lifespan. In rcm1 -knockout cells, however, already under unstressed, basal conditions a high extent of translational regulation of gene expression genes was observed, suggesting that the lack of C methylation specializes the ribosome complement for recruitment of different mRNAs out of the offered pool of totally transcribed mRNAs.

Remarkably, of those genes in unstressed rcm1 -knockout cells, only 79 genes were similarly regulated in unstressed wt cells Fig. Thus, one-third of all genes that are translationally regulated by H 2 O 2 in wt cells, are already differentially expressed by rcm1 deletion.

Counterintuitively, under these conditions also, gene sets associated with ribosome biogenesis were strongly and significantly overexpressed.

To further investigate this surprising finding, we compared stressed wt cells and rcm1 knockouts with unstressed wt cells on transcriptome and translatome level individually. We found that ribosome biogenesis was strongly downregulated both on transcriptome and on translatome level on stress and loss of Rcm1. Therefore, we conclude that both on rcm1 knockout as well as under oxidative stress, reduced levels of mRNAs of genes involved in ribosome biogenesis are present, but more efficiently translated.

This mechanism would allow the cells to faster modulate ribosome biogenesis and thereby also protein synthesis in the event of stress. Taken together, our translational profiling data suggest that the loss of rcm1 leads to a more efficient translation of stress-responsive mRNAs, even under unstressed conditions.

Thus, preactivation of the translational stress response by rcm1 deficiency and the resulting improved modulation of cytoplasmic translation on stress, might contribute to the increased stress resistance and longevity of the NSUN5-deficient model organisms studied Fig.

Interestingly, we also found that both on transcriptional and translational level, gene sets associated with RNA methylation and maturation were always significantly downregulated in rcm1 -knockout cells in comparison with their respective wt controls.

This suggests a feedback loop, in which the loss of a single-RNA-modifying enzyme Rcm1 induces a global downregulation of RNA processing. We confirmed the microarray results by comparing the expression values from a subset of genes from the array, including the top regulated genes previously published by Gerashchenko et al.

Here we describe that the loss of NSUN5 is a conserved mechanism increasing the lifespan and stress resistance of yeast, flies and worms, which is mediated by the lack of methylation of a single rRNA nucleotide. This is so far one of the rare examples of direct physiological effects of a ribosomal RNA modification. Although a point mutation of C of 25S rRNA, which makes methylation impossible, is sufficient to confer an increased chronological lifespan in yeast Fig.

Thus, the identification of additional NSUN5 methyltransferase target sites by recently developed techniques, such as Aza-IP 35 , might be of great interest. Remarkably, reduced levels of NSUN5 as assessed by polysome profile analysis did not decrease bulk protein translation Fig. However, a specific protein, namely, the overexpressed luciferase reporter protein without a PTC was less efficiently translated on loss of Rcm1, as well as under oxidative stress Fig.

Although luciferase is no endogenous yeast gene, this might indicate that specific proteins, such as the PTC-less luciferase reporter, are differentially translated.

Even if the reporter is ectopically overexpressed and thereby might overload the translational machinery, this finding would be in line with the observation that loss of NSUN5, as well as oxidative stress, specifically control recruitment of stress-responsive mRNAs into polysomes compare Fig.

While an upregulation of cell cycle control proteins arrests the cell to avoid propagation of damage during DNA synthesis and mitosis, the increased amounts of DNA damage repair factors and regulators might help to sense and repair diverse lesions, before resuming the cell cycle.

Our results on the differential recruitment of stress-responsive mRNAs support the concept of specialized ribosomes. First, although oxidative stress induces substantial translational reprogramming of gene expression both in wt and rcm1 -knockout cells, there is a drastic difference in the pattern of recruited mRNAs between the two genotypes. Thus, we propose that the unique oxidative stress response on loss of rcm1 arises from the presence of a cellular complement of redox-sensitive specialized ribosomes that carry unmethylated C Second, the expression of 23 genes is translationally regulated under every condition tested and we therefore conclude that this subset represents differentially translated proteins necessary for obligatory cellular functions, regardless of the genotype or stress.

This small overlap documents that only a minor fraction of the overall translational stress response acts independently of Rcm1 function and further emphasizes that the Rcm1-mediated response is distinct from that of wt cells.

Further analysis of these data sets to clarify the individual roles of selectively expressed stress-responsive proteins will be presented elsewhere. Since NSUN5 transcription was found to decrease in cellular model systems during aging under physiological conditions, as a consequence lower levels of C methylation will favour more efficient translation of stress-response proteins.

Elevated levels of these might consequently enable cells to better tolerate oxidative stress and prolong lifespan. Alternatively, this might be part of a stress-defence memory of cells that have been repeatedly exposed to stress during their lifespan and will therefore be more resistant during consecutive encounters and thus live longer.

However, we cannot exclude the possibility that lack of NSUN5 poses a beneficial stress to the cells and thus indirectly increases the recruitment of stress-related mRNAs. By any of these depicted mechanisms, knockdown of NSUN5 extends lifespan and stress resistance without affecting body size or fecundity of flies and worms, while most of the known genetic and nutritional interventions extending the lifespan of organisms also antagonistically decrease growth, body size and fecundity 10 , Thus, it remains elusive why evolution selected for the presence of NSUN5, which shortens lifespan on reduced nutrient availability and has so far no observable beneficial effects.

It seems likely that the loss of NSUN5 also has detrimental effects, for example, the reduced replicative lifespan of yeast mother cells, the reduced translational fidelity, or other not yet identified ones, which appear only under certain environmental conditions.

Probably, these detrimental effects counteract beneficial ones and are neutralized by mild dietary restriction, which leads to the observed lifespan extension only under reduced nutrient availability. Our observation that NSUN5-mediated lifespan extension is dependent on lower amounts of food, or food with lower energy content, might also be explained by a putative link between NSUN-5 activity and nutrient availability through nutrient sensing pathways, such as TOR, or sams Therefore, further studies aiming at the identification of upstream regulators of NSUN5 activity, especially in response to dietary conditions, are required to fully understand the biological function of NSUN5 and significance of C methylation, which led to the high evolutionary conservation of both.

Our finding that cell cycle control proteins are translationally upregulated on rcm1 knockout in exponentially growing yeast cells suggests that loss of Rcm1 induces a translational response, which might vary depending on the growth phase.

Furthermore, the reduction of replicative lifespan might be a consequence of the activation of proteins involved in the negative regulation of the cell cycle. Thus, our proposed molecular mechanism underlying NSUN5-mediated stress resistance and longevity, which we conclusively tested in replicating yeast, needs to be validated in non-replicating yeast.

However, ribosomal profiles from stationary phase cells closely resemble those of oxidatively stressed wt cells personal communication L. Moreover, translational fidelity and mRNA recruitment are also altered by entry into stationary phase personal communication L.

Analysis of this matter in detailed genetic and biochemical assays is needed and will be the scope of further studies, as well as testing our proposed molecular mechanism in C.

This might prove to be technically demanding, since methods for testing ribosome conformation and fidelity are not yet well established in these organisms. Finally, our data together with previous reports suggest that the cellular reaction to various signals, such as nutrient availability 3 , oxidative stress 13 , 15 , 17 and heat shock 14 , converge on changing ribosomes not only in terms of ribosomal proteins, their modification or stoichiometry, but also in terms of rRNA modifications.

In the case of NSUN5, this change probably occurs already during ribosome biogenesis, since Rcm1 was reported to localize to the nucleoli 28 , 29 , where rRNA is transcribed and processed and preribosomal particles are assembled Thus, the NSUN5-mediated stress response is probably slow, since it depends on the exchange of methylated with unmethylated ribosomes. This leads us to propose that NSUN5 activity is preferentially modulated by long-term or chronic stress, as during the aging process.

These specialized ribosomes have the unique ability to sense stress and consequently alter their structural confirmation, as well as fidelity. This enables them to selectively recruit and translate mRNAs to react to distinct cellular signals, such as various stressors, in a fast and efficient way 18 , 19 , In summary, we propose that NSUN5 contributes to the generation of stress-counteracting specialized ribosomes, which participate in extending the life- and healthspan of cells and organisms.

It is tempting to speculate how stress-counteracting ribosomes might be employed to improve health conditions of an aging society. Sample sizes were estimated from literature for the respective assays.

No systematic randomization method was used, but samples were never prepared and analysed in a predefined order. No systematic blinding throughout all assays was done, but at least one replicate of all lifespan- and stress resistance assays was performed with similar outcome by a different operator, who was blinded to the group allocation.

The sequences for shRNAs are as follows:. The control flies were the progeny of the cross between the lines used to create the transgenic lines w for the RNAi lines and y[1] w[67c23] for the lines overexpressing dNsun5 and the GAL4 driver carrying both the transgenic and GAL4 constructs.

All flies used in experiments were mated once and collected soon after emergence unless specified otherwise. Flies were kept on a 2. Figures show representative data of two independent biological replicates. Flies were collected and placed in plastic vials, 20 males in each vial.

Around flies were used in each group for each replicate. Dead flies were counted every weekday until the death of the last fly. They were transferred to fresh food twice a week. The Kaplan—Meier survival curves were computed and compared with log-rank tests implement in SigmaPlot version 11 Systat Software.

Lifespan measurements were carried out on both RNAi flies and flies overexpressing dNsun5. Twenty-one-week-old males were transferred into a sealed vial in which a paper filter soaked with water had been inserted for a total of flies per group and replicate. Ten males and 10 females per group control and dNsun5 RNAi were anaesthetized under light CO 2 and lined up under a stereomicroscope, upwards facing.

Pictures of the thorax were taken, all at the same magnification, and the length of the thorax was measured from the pictures length expressed in number of pixels.

Forty males were collected for each genotype with 10 flies per vial. At 12, 19, 26 and 47 days of age, flies were transferred into empty vials and their climbing activity was measured in two different ways:.

First, each vial was placed on a shaker and a gentle shake was given for all the flies to fall to the bottom of the vial. The measurement was repeated three times for each vial. The percentage of flies able to reach the top of the vial was computed and the effect of age and genotype was assessed with a two-way ANOVA with Bonferroni post tests using GraphPad Prism version 5. The height reached by each fly was then recorded. In this measurement, the effect of age and genotype on the distance climbed by each group was tested with a two-way ANOVA with Bonferroni post tests as before.

Twenty virgin females were collected for each genotype and put singly into vials with two males of the same genotype. Flies were transferred into new vials three times a week and the vials were checked for dead flies. Strains were cultured under standard laboratory conditions on E.