We are interested in the biology of the G0-like resting states that eukaryotic cells enter when conditions are not conducive to continued growth. Our goal is to define the processes that are induced during these periods of quiescence and to determine how they collectively contribute to cell survival. This proposal extends this analysis and examines a particular ribonucleoprotein (RNP) complex that forms in the cytoplasm of resting cells, known as a Processing-body, or P-body. P-bodies are relatively large aggregate structures that contain non-translating mRNAs and a distinct set of protein constituents, including a number of enzymes involved in the processing of these transcripts. Interestingly, the P-body is just one of a large number of similar cytoplasmic structures that form in the G0 cell. This prevalence suggests that these complexes are important for the biology of the resting cell, but little is presently known about the underlying reasons for this large-scale sequestration of macromolecules. These cytoplasmic structures also appear to be important for human health as a related RNP complex, the stress granule, has been implicated in the pathology of neurological disorders, like amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type 2. In all, thes data suggest that these cytoplasmic foci play important and diverse roles in eukaryotic biology. It is therefore critical that we develop a better understanding of the functions of these structure and the manner in which their assembly is regulated. This proposal addresses these broader issues by examining a model RNP complex, the P-body of the yeast, Saccharomyces cerevisiae. P-bodies have been conserved from yeast to humans, and much of what we know about these RNP structures has come from studies with this budding yeast. However, despite extensive effort, little was known about the mechanisms regulating P-body assembly and the physiological role of the larger foci that form in quiescent cells. Our recent work has suggested interesting answers to both of these questions. In particular, we have identified the cAMP-dependent protein kinase (PKA) as a key regulator of P- body assembly, and found that the larger P-body foci appear to be required for the long-term survival of quiescent cells. Our data indicate that PKA directly phosphorylates Pat1, a conserved core constituent of P- bodies, and thereby disrupts the formation of the larger aggregate structures. The experiments proposed here aim to define the mechanistic details of this control by PKA and to elucidate how the larger foci promote cell viability. Finally, we will explore further the intriguing observation that signaing molecules, like PKA itself, are also found in P-bodies and related complexes. The underlying hypothesis to be tested is that specific proteins and mRNAs required for the subsequent resumption of growth are stored and protected within P-body foci in quiescent cells.
The specific aims of this proposal are: (1) to determine the role of PKA signaling in the regulation of P-body foci formation; and (2) to determine the physiological role of P-body foci in quiescent cells.
This proposal examines the biology of cytoplasmic structures that have been implicated in the pathology of several neurological disorders, including amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type-2. By increasing our understanding of the general mechanisms regulating the assembly of these ribonucleoprotein complexes, the work here could help inform efforts aimed at the treatment of these conditions.
