Post-transcriptional gene silencing mediated by microRNAs (miRNAs) is an established paradigm in metazoan gene regulation, playing critical roles in a wide array of biological processes including developmental timing, organogenesis, and cellular differentiation and proliferation. Mechanisms governing miRNA biogenesis and target silencing are largely established; however, a significant deficit remains in our understanding of how post- translational modifications of the miRNA-mediated silencing complex (miRISC) affect its stability and integrity, subcellular distribution, and effector functio in repressing target mRNA expression. Our long-term goal is to understand how post-translational modifications of miRISC influence miRNA-mediated gene silencing. The specific objective of this proposal is to investigate a novel role for protein kinase CK2 in the miRNA pathway through phosphorylation of core miRISC subunits. Our preliminary data indicate that CK2 physically interacts with miRISC through phosphorylation of two miRISC components: the DEAD-box helicase CGH-1 and the vasa intronic gene product VIG-1. Based on our preliminary data, we hypothesize that through phosphorylation of CGH-1 and VIG-1, CK2 plays a critical role in promoting miRISC binding to target mRNAs. We will test our hypothesis by pursuing two specific aims: 1) Evaluate the key role of CGH-1 and VIG-1 phosphorylation in the specificity of miRNA target binding and expression and their biological significance during animal development. 2) Define the role of CK2 activity and miRISC phosphorylation on the compositional integrity of miRISC and its recruitment to the miRNA target degradation centers, the P bodies. In addition, we will explore potential regulation of CK2 activity by the opposing function of candidate PP2A phosphatases on the phosphorylation state of miRISC. The proposed research is significant because it will establish post-translational modification of miRISC as a general mechanism required for miRISC assembly, localization, and silencing function that impacts all biological pathways regulated by miRNA-mediated gene silencing. The basic insights obtained will also inform the rapidly advancing field of miRNA therapeutics for treating complex human diseases.
MicroRNA-mediated gene silencing is critical for human development and dysregulated in a variety of complex disorders including metabolic diseases, neuropathies, and cancer. Using the model organism C. elegans, where the first microRNAs were discovered, we will address a major gap in our understanding of how post-translational modifications of the core silencing complex, termed miRISC, affect microRNA-mediated gene expression. We anticipate that our proposed research will advance not only our basic understanding of microRNA-dependent mechanisms, but also provide insights into new therapies designed to modulate aberrant gene expression in complex human diseases with the ultimate goal of improving human health.
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