UC San Diego

Aging, an inevitable path of life defined by gradual deterioration of function and ultimately death. While young cells and organisms are generally able to maintain health and homeostasis how cells lose this ability over time remains a great biological question. A common theme throughout this thesis will be the networked interaction of cellular processes and how an aging landscape can shape these interactions and drive cellular aging fates. In Chapter 1, we take a deep analysis of the molecular interactions that govern aging with a specific focus on what drives changes cellular proteostasis as cells age. We assess the knowledge regarding what governs the cellular proteostasis network and what factors change with age that drive a loss of protein homeostasis. We take particular interest in RNA binding proteins as these proteins are highly prone to aggregation and they have been widely implicated in many age-related and neurodegenerative disorders. We further analyze data about specific types of RBPs and the implications that their aggregation has on cellular aging and aging pathologies. We highlight the role of the nucleolus and rRNA binding proteins in regulating cellular and protein homeostasis, as we hypothesize that increase rRNA transcription is a major driver of loss of nucleolar stability, and loss of cellular protein homeostasis. One of the ways rRNA transcription is regulated is by chromatin silencing and we posit that increased chromatin stability via Sirtuins and other chromatin modifiers may increase nucleolar stability and protein homeostasis thereby increasing cellular longevity. In Chapter 2, we perform a systematic study of ribosomal RNA-binding proteins and uncover an interesting link between chromatin stability and loss of protein homeostasis. Using time-lapse microscopy and microfluidics we discover differences in cellular protein homeostasis within populations of isogenic cells and discover that this divergence is consistent with two distinct aging modes. The two distinct aging modes are characterized by changes in daughter cell morphology and by changes in Sir2 activity and hence rDNA silencing activity. “Mode 1” aging cells are characterized by elongated daughter aging morphology and dramatic loss of rDNA silencing whereas “Mode 2” cells are characterized by small, rounded daughter aging morphology and maintained rDNA silencing. We provide evidence that not only is there a correlation between cells that lose rDNA silencing and cells that have proteostatic stress, but also that accompanying ERC formation and elevated rDNA transcription drives loss of proteostasis. Next, we systematically investigate RNA binding proteins, which are among the most aggregation prone cellular proteins, to see if and which of these proteins aggregate in response to loss of Sir2 activity. We perform a high-throughput screen of nearly 150 RBPs and identify 27 RBPs that aggregate in response to Sir2 inhibition. We find the most enriched sub-category within the hits are ribosomal RNA binding proteins. Given the link between Sir2, rDNA, and rRNA binding proteins we chose to systematically assess the aggregation of each of these genes during aging. Here we find that, consistent with general proteostasis decline, that Mode 1 cells exhibit aggregation of these rRNA binding proteins whereas Mode 2 cells do not. We hypothesize that elevated rRNA transcription that occurs in Mode 1 cells helps drive the nucleolar dysfunction and aggregation of these rRNA binding proteins. We provide evidence that elevated rRNA transcription does indeed increase rRNA binding protein aggregation and cellular protein aggregation more broadly. Finally, we demonstrate the lifespan impacts that these rRNA binding proteins have, illustrating the negative impacts that rRNA binding protein aggregation has on cellular lifespan.