Molecular mechanisms of translational regulation in aging Genome-wide microarray and RNA sequencing studies have revealed changes in the expression of hundreds of genes during aging in diverse organisms. Transcriptional regulation plays an important role in the control of gene expression during aging; however, translation efficiency likely plays an equally important role in determining protein abundance, but has been understudied in this context. Here we propose to study translational changes that are associated with increased longevity induced by dietary and genetic interventions and examine the mech- anisms of post-transcriptional gene regulation in aging using yeast as a model system. We will test the hypoth- esis that, in response to dietary restriction (DR) and genetic alterations that extend lifespan, mRNA-binding pro- teins (RBPs) coordinately regulate diverse cytoprotective genes by affecting their translation efficiency. To iden- tify RBPs involved in regulation of these processes, we will apply RNA-Seq and ribosome profiling combined with next-generation sequencing and characterize transcriptional and translational changes in replicatively aged yeast cells in response to DR and in a panel of long-lived gene deletion mutants identified in genome-wide screens. Using the Bayesian network modeling approach, we will integrate transcriptional and translational pro- filing data with information about transcription factor (TF) binding sites and sequence elements recognized by RBPs and build a regulatory interaction network. We will then characterize RBPs and directly identify their mRNA-binding targets. These data will allow us to uncover specific mechanisms and identify cis-regulatory ele- ments that are responsible for the translational signatures associated with increased longevity. We will also utilize fluorescence microscopy and cutting-edge microfluidic technologies in order to monitor how the abun- dance of RBPs changes with age at the single-cell level. Finally, we will use reverse engineering approach to test if the regulators identified using our systems approach play a causative role in mediating the lifespan exten- sion through genetic epistasis experiments. Successful completion of this study will add valuable insight into fundamental principles of translational regulation, and may provide a better understanding of the molecular mechanisms of aging and lifespan control.

Project Narrative The major goal of this grant application is to examine the mechanistic basis of post-transcriptional gene regula- tion in aging using yeast as a model system. For this, we propose to quantitatively analyze transcriptional and translational changes in replicatively aged cells in response to dietary and genetic interventions known to extend lifespan. This information will be integrated to develop a regulatory interaction network and identify factors in- volved in translational regulation of aging genetic pathways.