Dietary restriction (DR) extends lifespan and has benefits for general health in a myriad of organisms ranging from single-celled yeast to mammals. This fact makes the mechanisms regulating DR attractive targets for developing therapeutics to improve health in humans. Despite its importance, there are many gaps in our understanding of the mechanisms governing the response to DR. In particular, the need to understand the effects of DR on post-transcriptional processing is exemplified by studies showing a lack of correlation between transcript abundance and protein expression. By combining polysomal (i.e. translated) mRNA isolation with RNA-Seq, gene expression changes that arise from altered mRNA translation can be resolved in a C. elegans model of DR. In a pilot study, we found changes in post-transcriptional processing centered around nonsense mediated decay (NMD) and alternative splicing that were associated with changes in mRNA translation. NMD detects and degrades aberrant mRNA, including forms produced by alternative splicing. Serine/arginine-rich (SR) proteins govern alternative splicing and even alternatively splice a portion of their own transcripts in a way that makes them subject to NMD. This self-regulating activity creates a negative feedback that limits SR expression. Preliminary studies show that DR lowers NMD and increases translation of SR proteins. Deeper analysis of the translatome data in the pilot study shows evidence of increased alternative splicing, including splicing of ribosomal mRNA to nonsense isoforms. This potentially explains diminished ribosome biogenesis previously observed under DR that could not be explained by transcription changes. Further testing shows that lowering expression of ribosomal mRNA with RNA interference increases lifespan and that factors involved in regulating NMD are required for increased lifespan under DR. This proposal tests the hypothesis that DR lowers NMD to increase mRNA translation of SR proteins, which promotes alternative splicing of ribosomal mRNA to nonsense-bearing isoforms, resulting in increased lifespan.
Aim 1 will establish the mechanism responsible for the downregulation of NMD under DR and confirm the role of NMD in regulating SR protein translation.
Aim 2 will investigate the requirement for alternative splicing and specific alternatively spliced ribosomal mRNA in increased lifespan under DR. By investigating the role of NMD and alternative splicing in longevity regulation by DR, we will establish novel biological roles for these post-transcriptional processes. Additionally, by determining the necessity of ribosomal protein isoforms for longevity, our work will promote future investigations into the role of ribosome composition in stress responses.
Dietary restriction without malnutrition is the most robust environmental condition to increase longevity and health in a myriad of organisms. The evolutionarily conserved response to dietary restriction suggests that what we learn could be targeted for therapeutic use in humans. This proposal is focused on how gene expression is coordinated post-transcriptionally to increase lifespan in response to dietary restriction.
