The eukaryotic cell is a highly compartmentalized structure that is subdivided into discrete functional areas by the presence of a variety of membrane-enclosed organelles. This segregation of functions is essential for normal cell growth and survival. Interestingly, recent studies have indicated that additional levels of compartmentalization exist within these cells. In particular, a number of cytoplasmic granules that contain distinct sets of proteins and mRNAs have been identified. Two of the best-characterized of these ribonucleoprotein (RNP) structures are the Processing-body (P-body) and stress granule. These granules differ from the more traditional organelles in that they lack a limiting membrane and are rather dynamic in nature. These granules are evolutionarily conserved and have been linked to a number of human diseases, including a variety of cancers and neurodegenerative disorders. However, despite these observations, the physiological functions of these RNP structures remain poorly understood. This lack of understanding represents a critical gap in our current knowledge and attempting to bridge this divide is a primary research focus in our lab. The experiments in this proposal aim to further our understanding of both the biological roles of these RNP granules and the mechanisms that regulate their assembly. The first two aims come at this question of biological function from different directions. In the first, we will assess the physiological consequences of having key signaling proteins associate with the P-body and/or stress granule during conditions of stress. Our focus here is on particular protein kinases and the possibility that this re-localization to RNP granules allows for a rewiring of the signaling networks present in the cell. In the second, we focus on the granule as a whole and ask how cell physiology is altered in mutants that lack these RNP structures. These latter studies focus on a potential role in protein homeostasis that was suggested by recent work from our lab. In particular, we have found that mutants lacking P-bodies exhibit elevated levels of protein misfolding and aggregation. The experiments here aim to determine the underlying mechanisms responsible for these effects and should establish whether these RNP granules have a direct role in the maintenance of protein quality control (PQC). Finally, the third aim examines several key aspects of the regulation and assembly of P-body foci. In particular, the studies will define the molecular mechanism underlying PKA-mediated control of P-body assembly and develop a facile method for the purification of these RNP granules. The three specific aims of the proposal are as follows: (1) determine the physiological consequences of protein kinase recruitment to RNP granules; (2) define the underlying mechanisms responsible for the P-body-mediated effects on PQC; and (3) examine the process and regulation of P-body assembly.
This proposal examines the biology of a family of cytoplasmic granules that have been implicated in the pathology of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type-2. The proposed studies aim to define how these structures are involved in these disorders and the possibility of using this information to develop novel therapeutics for these conditions.
