The goal of this research is to elucidate the mechanisms that govern mRNA degradation. The principal pathway for mRNA degradation in yeast involves an initial poly(A) deadenylation, followed by 5'end decapping, and 5'-3'RNase digestion. The rate of deadenylation of different mRNA varies considerably with corresponding effects on the rate of degradation of the RNA. The major cytoplasmic deadenylase in yeast is the CCR4-NOT complex, and CCR4's ability to deadenylate is dependent on the state of the mRNP structure. The mRNP consists of the poly(A) binding protein (PAB1) bound to the poly(A) tail, translation initiation factors (TIFs) linking the 5'end of the mRNA to the 3'end of the RNA, and other factors, such as PUF3, that bind to the 3'UTR of mRNA. The PAB1-mRNP plays a key role in regulating deadenylation. The proline-rich (P) and RRM1 domains are required for CCR4 deadenylation and mediate PAB1 self-association;the P domain also appears to affect PAB1's off rate with the poly(A). PAB1 self-association, in turn, inhibits the ability of PAB1 to bind poly(A), suggesting that factors affecting PAB1's off rate and/or self-association will control CCR4 action. We have also established that TIF defects accelerate deadenylation dependent on these two domains of PAB1, and the TIF, eIF4G, make contacts to the RRM1 and P domain. Moreover, we have found that the UPF1 protein, which accelerates nonsense-mediated deadenylation, binds specifically to RRM1 of PAB1 and is required by this domain to accelerate CCR4 action. Similarly, PUF3 accelerates deadenylation dependent on the RRM1 domain of PAB1. In this proposal, we will address how PAB1 inhibits deadenylation and what factors are involved in removing PAB1 from the mRNA to allow CCR4 function. The effects of RRM1 PAB1 mutations on the off-rate of PAB1 will be determined and correlated to their effects on CCR4 in vivo deadenylation and PAB1 self- association. In vitro systems will be developed to examine the effects of RRM1 mutations on CCR4 deadenylation, and analytical ultracentrifugation will be used to study PAB1 self-association. The effects of PUF3/UPF1/TIFs on controlling PAB1 interaction with the poly(A) tail, on PAB1 self-association, and on CCR4 deadenylation in vitro will be assessed. PUBLIC HEALTH RELEVANCE: This proposal is relevant to public health by studying how protein expression is controlled. The characterization of the factors that regulate when and to what extent proteins are synthesized will illuminate the processes by which aberrant protein production leads to particular disease states.