Degradation of cytoplasmic mRNA plays an essential role in the regulation of gene expression and in quality control of mRNA biogenesis. Nearly all eukaryotic mRNA decay pathways are initiated by deadenylation, i.e., the shortening of the 3' poly(A) tail, which has two distinct phases in mammalian cells. One major consequence of deadenylation is the formation of non-translatable mRNA-protein complexes (mRNPs), which leads to mRNA decay or translation silencing. Deadenylation is often a rate-limiting step for mRNA decay and translational silencing, making it an important point for controlling the function and fate of cytoplasmic mRNA. However, presently little is known about how this fundamental process is regulated in mammalian cells. Particularly, it is unclear what kind of mRNP remodeling occurs during each of the two phases of deadenylation and how individual mRNP remodeling steps impact the fate of cytoplasmic mRNA in general. In the past funding period, we have made several key findings that lay the groundwork for addressing these important questions in mammalian cells. We discovered that: 1) miRISC complex triggers rapid mRNA decay by recruiting both Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes to accelerate the first and second phases, respectively, of target mRNA deadenylation; 2) the first phase of deadenylation is coordinated by two functionally distinct isoforms of Pan3, a regulatory factor of Pan2 deadenylase; 3) reversible phosphorylation in intrinsically disordered regions (IDRs) of TNRC6 (an effector for micro mRNA (miRNA)-mediated gene silencing) and of Pan3 modulates the interactions of these proteins with cytoplasmic poly(A)-binding protein (PABP)C1 and/or deadenylases, which in turn controls their cellular functions; and 4) TNF and ROCK kinase inhibitors have negative and positive effects, respectively, on miRNA-mediated gene silencing. In the current proposal, we will: 1) determine the basis for the distinct actions of the Pan3L and Pan3S isoforms in the regulation of mRNA deadenylation and the role of phosphorylation in the Pan3 IDRs in the process; 2) elucidate the biological significance of the biphasic nature of deadenylation and the details of mRNP remodeling that occur in each phase of deadenylation; and 3) elucidate novel mechanisms by which miRNA- mediated mRNA decay is regulated globally via modulating the ability of miRISC complexes to promote deadenylation. The proposed studies will provide a mechanistic framework for understanding dynamic and signal-dependent control of cytoplasmic mRNA functions via intrinsically disordered proteins. They also represent a meaningful advance towards our long-term goal of understanding the biological principles and mechanisms that govern cytoplasmic mRNA metabolism in the context of mRNPs in mammalian cells.
The proposed studies will reveal fundamental principles that govern mammalian messenger RNA turnover and protein synthesis. Since many human diseases (e.g., cancers, autoimmune diseases, allergic inflammation, etc.) are associated with changes in gene expression due to uncontrolled messenger RNA functions, the proposed study has the potential to unravel new mechanisms underlying these conditions and thus facilitate the development of novel therapeutic agents.
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