Genome-wide microarray and RNA sequencing studies have revealed changes in the expression of hundreds of genes during aging in diverse organisms. Transcriptional regulation clearly 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 woefully understudied in this context. Here we propose to study translational changes that are associated with increased longevity and examine the mechanisms of post- transcriptional gene regulation in aging using yeast as a model system. We will test the hypothesis that, in re- sponse to genetic alterations that extend lifespan, mRNA-binding proteins (RBPs) coordinately regulate di- verse cytoprotective genes by affecting their translation efficiency. To identify 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 a panel of long-lived gene deletion mutants identi- fied in genome-wide screens. We propose to integrate translational profiling data obtained for long-lived mu- tants with information about structural and sequence elements recognized by RBPs and build a regulatory in- teraction network. We also propose to carry out ribosome profiling in replicatively aged wild-type cells and long-lived mutant strains to globally identify genes whose expression is affected by translational regulation dur- ing aging. Finally, we will utilize cutting-edge microfluidic technologies to validate and extend these discover- ies at the single-cell level. Comparing translational profiles in young and replicatively aged wild-type yeast and multiple long-lived deletion mutants will reveal genetic signatures associated with increased longevity and will allow us to identify novel RBPs involved in translational regulation during aging. We will then characterize RBPs and directly identify their mRNA-binding targets using CLIP-Seq. These data will allow us to uncover specific mechanisms and identify cis-regulatory elements that are responsible for translational changes ob- served in long-lived mutants. We will also use fluorescence microscopy and microfluidic cell trapping in order to monitor how the abundance of RBPs changes with age in individual mother cells. Finally, we will test if the candidate RBPs identified from CLIP-Seq and microfluidics experiments play a causal role in mediating the lifespan extension through genetic epistasis analysis in order to determine whether candidate RBPs are nec- essary and sufficient for lifespan extension. Successful completion of this study will add valuable insight into translational regulation of aging, and may provide a better understanding of the molecular mechanisms that regulate aging in humans.

PUBLIC HEALTH RELEVANCE STATEMENT: The major goal of this grant application is to examine the mechanistic basis of post-transcriptional gene regulation in aging using yeast as a model system. For this, we propose to quantitatively analyze transcriptional and translational changes in a panel of long-lived single-gene deletion mutants and identify the signatures associated with increased longevity. This information will be inte- grated to develop a regulatory interaction network and identify factors involved in translational regulation of ag- ing genetic pathways.