The overall objective of this proposal is to determine the role of stress granules (SGs) and processing bodies (PBs) in reprogramming mRNA metabolism during stress. SGs are cytoplasmic foci at which translationally stalled pre-initiation complexes accumulate in stressed cells. We have discovered that SGs are in physical proximity to PBs, distinct cytoplasmic foci at which decapping enzymes, exonucleases and partially degraded mRNAs are concentrated in both stressed and unstressed cells. Moreover, components of the RNA-induced silencing complex (RISC) are concentrated at both SGs and PBs, suggesting that microRNA may be involved in stress-induced reprogramming of mRNA metabolism. We hypothesize that mRNA released from disassembled polysomes is sorted and remodeled at SGs, allowing the delivery of selected transcripts to PBs for degradation. The ability of the mRNA destabilizing factor TTP to cause SGs and PBs to fuse into a common structure supports this hypothesis and suggests that TTP serves as a molecular link between SGs and PBs.
The specific aims are: i) To determine whether mRNA and protein move between SGs and PBs, ii) To determine the contribution of SGs, PBs and TTP to stress-induced reprogramming of mRNA metabolism, iii) To determine the role of RISC in stress-induced reprogramming of mRNA metabolism, and iv) To determine the role of mTOR and S6 kinases in SG/PB assembly and stress-induced reprogramming of mRNA metabolism.
These aims will be accomplished by using fluorescence recovery after photobleaching analysis to quantify movement of mRNA and proteins in and out of SGs and PBs. We will compare the ability of mutant and wild type TTP to regulate both SG:PB interactions and mRNA metabolism. We will use reporter transcripts that are regulated by microRNA to determine whether the RISC regulates mRNA metabolism during stress. Finally, we will determine whether a mTOR/S6 kinase cascade that regulates the assembly of SGs is also involved in the regulation of mRNA metabolism during stress. These studies have implications for our understanding of stress-regulated pathological conditions, including Alzheimer's disease, diabetes and cancer.
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