Un nouveau gène de la longévité ouvre de nouvelles pistes pour vieillir mieux. L'accroissement de la longévité induit par la suppression des tissus reproducteurs a été observé chez la drosophile et chez le ver. Chez ce dernier, l'opération lui donne 60% de vie en plus et lui permet un vieillissement harmonieux et en bonne santé. Les mécanismes moléculaires qui induisent cette réponse font l'objet d'intenses recherches. Certains gènes étaient déjà connus pour être associés à l'accroissement de la longévité des vers sans lignée germinale, et nous avons démontré l'existence d'une nouvelle voie impliquant le récepteur nucléaire NHR-80. Les nématodes dépourvus de lignée germinale et dont nhr-80 est muté ne voient pas leur longévité augmenter. En outre, la surexpression du gène allonge davantage leur durée de vie: elle est 150% plus longue que celle de leurs congénères sauvages. Cela démontre l'importance de ce récepteur nucléaire dont l'activation par une hormone encore inconnue enclenche l'expression ou la mise sous silence de centaines d'autres gènes. Notamment, nous avons montré que l'une des cibles de NHR-80, l'enzyme FAT-6 qui transforme l'acide stéarique en acide oléique est fondamentale, puisque les vers dépourvus de lignée germinale ne présentent plus aucun gain en longévité en l'absence de FAT-6. À terme, nous espérons pouvoir récapituler les effets de l'ablation de la lignée germinale chez un organisme fertile, c'est à dire, d'induire les réarrangements métaboliques qui ont lieu suite à cette opération afin d'en tirer les effets positifs sur la santé, sans affecter la reproduction. / Discovery of a key longevity gene opens new perspectives for healthy aging.Increased longevity induced by reproductive tissues removal (germline ablation) is observed in the fly Drosophila melanogaster and in the worm Caenorhabditis elegans. In the latter, the operation increases lifespan by 60%, and enables the nematode to age harmoniously and in good health. The molecular mechanisms that induce this response are subject of intensive research. Our study reveals the existence of a new powerful longevity gene, nhr-80, which mediates this longevity effect. We have shown that inactivation of nhr-80 prevents lifespan increase. Furthermore, nhr-80 overexpression lengthens the nematodes' lifespan by 150%! nhr-80 encodes a nuclear receptor, which activation by a still unknown hormone controls the expression of hundreds of other genes. We showed that one of the critical NHR-80 targets, the enzyme FAT-6, which transforms stearic acid into oleic acid, is necessary to prolong lifespan since a mutation of the fat-6 gene suppresses the effects of germline ablation on longevity. The next step will be to determine how an increase in the level of oleic acid induces an adaptive response resulting in increased longevity. This research may lead to the exciting possibility of recapitulating the benefits of germline ablation in fertile animals; in other words, to activate the longevity effects normally triggered by germline ablation in order to fight, in one go, a host of diseases associated with aging, without affecting reproduction.

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published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

In all organisms studied so far, the recognition of a functional poly(A) site is essential for RNA polymerase II termination at the end of nearly all genes transcribed by this enzyme (Whitelaw and Proudfoot, 1986; Guo et al., 1995; Birse et al. 1997). A number of eukaryotes have some of their genes organised in polycistronic structures which resemble bacterial operons (Davis and Hodgson, 1997; Ganot et al., 2004; Spieth et al. 1993), and in C. elegans, approximately 20% of all genes are contained within these operon-like structures (Blumenthal et al., 2002). Here, functional poly(A) sites will be synthesised and recognised by RNA polymerase II at the end of each gene within the operon, however termination of the polymerase only occurs at the final gene of the polycistronic transcription unit In these studies, we analyse the halting of RN A polymerase II transcription at the end of monocistronic genes and furthermore observe how premature RNA polymerase II termination is prevented during polycistronic transcription in the nematode C. elegans. We predominantly make use of reverse transcriptase PCR-based techniques to examine these mechanisms. We show that a large increase in pre-mRNAs stretching into the 3' flank of genes can be detected in worms depleted of the riboexonuclease XRN-2, indicating that this enzyme may have a possible role in RNA pol II termination and 3' end formation in C. elegans. Furthermore, we provide evidence that the polymerase can read into telomeric structures in the nematode. Also, we demonstrate that an RNAi-mediated knockdown of the UI-70K subunit of the UI snRNP causes a drop in polycistronic transcripts, providing a link between cis- splicing and the prevention of premature RNA polymerase II termination at operon-internal poly(A) sites. Finally, we illustrate that operon-internal poly(A) sites are capable of directing efficient 3' end formation outside of a polycistronic background. Together, these findings provide valuable insights into the mechanisms involved in directing or preventing premature RNA polymerase II transcription termination at C. elegans poly(A) sites.

The diet an organism keeps is crucial in sustaining its health and fitness. The fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans are excellent models for nutritional studies due to their small size, large progeny numbers, quick development, and modifiable laboratory diets. Here I examine these two organisms in order to better understand the complex interrelationship between an animal and its diet. Previous work has shown that in the wild numerous organisms are capable of selecting specific nutrients in a non-random manner in order to maximize fitness. However, the genetic underpinnings driving these nutrient choices remain elusive. Female fruit flies consume higher levels of protein following mating to prepare for the costs of reproduction. I examined the role of S6 Kinase (S6K), a downstream effector of the nutrient-responsive target of rapamycin pathway, in mediating this decision. I demonstrate that neuronal S6K activity and serotonin are involved in regulating protein consumption when allowed to choose nutrients freely as well as following macronutrient deprivation; suggesting that they may play a role in mediating postmating dietary switch and maintaining nutrient balance. Modulating levels of dietary components can have extensive impacts on processes such as development, fecundity, and metabolism in multiple organisms. However, the influence of dietary genetics on the consumer is virtually unknown. I performed a screen feeding single-gene mutants of E. coli to C. elegans and monitored the effects on the insulin-like signalling pathway (ILS). When mutated, genes involved in multiple processes and functions in E. coli enhanced activity of the ILS downstream transcription factor, DAF-16. One mutant strain of E. coli I pursued had a knockout of the cAMP-producing, adenylate cyclase gene. Addition of exogenous cAMP to the diet containing live, metabolically active E. coli rescued all the effects of the mutant on C. elegans; thereby suggesting that bacterial metabolism of dietary cAMP can influence the C. elegans ILS. Collectively, my work demonstrates how the nutrient-sensing pathways of the consumer can shape and be shaped by interactions with its diet. These studies contribute to a better understanding of the consumer-diet relationship, and could help guide future work to investigate the role of diet in disease, quality of life, and longevity.

Mutation is a fundamental process that drives evolutionary change; however, most new mutations are deleterious for organismal fitness and can readily propagate within populations under a broad range of conditions. Mutational processes able to counteract deleterious mutation accumulation include: 1) reversion mutation back to wildtype, 2) acquisition of generally beneficial mutations, and 3) compensatory mutations that specifically mitigate the effects of previously-acquired deleterious mutations through epistasis. The potential for any of these mutation types alters our expectations for the impact of deleterious mutation in populations, but since the fitness effects of individual mutations are rarely characterized, the relative importance of beneficial and compensatory epistatic mutations is unknown. In this thesis, I characterized the nuclear mutations that arose in a previous mutation accumulation (MA) experiment using Caenorhabditis elegans nematodes, in which mutations were allowed to accumulate under extreme drift conditions in replicate, independently evolving lines initiated from a low-fitness mitochondrial electron transport chain (ETC) mutant, gas-1. In contrast to the results of typical MA experiments, gas-1 MA lines improved fitness slightly compared to their ancestor. Here, I find that the gas-1 MA lines demonstrate little increase in among-line variance and that the gas-1 MA nuclear mutations are more narrowly functionally defined than wildtype MA nuclear mutations. When combined with evidence for zygotic or post zygotic selection these data suggest that selection--both purifying and positive--can be an extremely powerful force even in conditions of extreme genetic drift. Furthermore, functional characterization of a four-mutation set isolated from one of the gas-1 MA lines on gas-1 and wildtype backgrounds shows fitness improvements on both backgrounds. This beneficial four-mutation set is associated with a decrease in steady-state endogenous ROS on the gas-1 background while exhibiting no effect on wildtype. I also find that steady-state ATP levels associated with the beneficial four-mutation set decreased compared to wildtype suggesting that fermentation may be metabolic strategy to cope with increase oxidative stress. These findings suggest that we can detect and characterize specific genetic changes that lead to a partial recovery of fitness and phenotype in a low-fitness ETC-deficient mutant strain of C. elegans. I extended my thesis to include analyses of fitness and phenotype of 24 replicate lineages of the gas-1 ETC mutant evolved in large population (n = 1000) sizes for 60 generations--conditions optimal for selection and fitness recovery (RC). I find that two distinct gas-1 RC fitness groups emerged: one group with significantly higher average fitness than the ancestor and containing two lines that exceeded wildtype fitness levels, and another group with more modest and non-significant fitness gains. Interestingly, many lines in the first group were observed to generate appreciable numbers of males during experimental evolution--consistent with evolution of outcrossing either accompanying or driving rapid fitness recovery. Bioinformatic functional analyses of the nuclear mutations that arose in the gas-1 RC lines show the availability of potentially more paths to fitness recovery for large populations than small ones. Combined, these data allow us to identify patterns in selection and drift in gas-1 recovery under MA and RC (recovery) conditions. My research advances our understanding of the genetic bases of adaptive evolution under extremely unfavorable population genetic conditions and how mitochondrial dysfunction affects evolutionary dynamics.

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