Project Summary RNA-binding proteins (RBPs) play key roles in regulating gene expression and many cellular functions. A lot of RBPs are aggregation-prone due to their low complexity, prion-like domains. While naturally-occurring aggregation of RBPs is important for the compartmentalized control of RNA metabolism, aberrant aggregation is detrimental and is associated with many diseases, in particular, age-related diseases such as neurodegenerative diseases and cancers. However, a systematic analysis of RBP aggregation and its functional consequences during aging remains lacking. Here we propose to conduct a systems biology analysis of age- dependent RBP aggregation using the replicative aging of S.cerevisiae as a model system. Our initial screen has identified positive RBP candidates that aggregate upon aging-related perturbations. Building upon these findings, we will combine innovative microfluidics with single-cell imaging technologies to systematically characterize these RBP aggregates during aging and to evaluate how these aggregates influence gene expression, aging phenotypes and the lifespan of individual living cells. In Aim 1, we will systematically characterize each of the identified RBPs that aggregate during aging. We will determine the biophysical and biochemical properties, material state and phase transition of RBP aggregates at different stages of the lifespan, which will provide important clues about how these aggregates influence cell physiology during aging. In Aim 2, we will investigate the interplay between RBP aggregation and cellular aging, focusing on how aggregation of RBPs is regulated by conserved aging-related pathways or factors, and how these aggregates contribute to age- dependent cellular changes and the final lifespan. In Aim 3, we will evaluate how RBP aggregation contributes to the proteomic changes during aging. We will use a newly-developed high-throughput microfluidic platform to identify target genes that are regulated by RBP aggregation and will examine their influences on aging, establishing the functional links among RBP aggregation, proteomic changes and aging phenotypes. Finally, we will integrate all the data generated in Aims 1, 2 and 3, delineate a systems-level regulatory network of RBP aggregation during yeast replicative aging, and develop a dedicated website for sharing the data to the scientific community. The RBP-regulated network will provide mechanistic insights into the causes, control and consequences of pathological RBP aggregation in aging and will be used to guide the design of new hypotheses and experiments, laying the foundation for the development of therapeutic and preventive strategies towards age-associated diseases.