Circadian regulation of microRNA biogenesis and function

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Regulation of mRNA translation in neurons is a critical point of control for processes of the developing and adult brain that require specific changes in gene expression. Emerging data indicate that a number of these processes - including synaptic plasticity, neurogenesis, and memory formation - are also regulated by circadian rhythm. We have obtained evidence for a mechanistic link between translational control and circadian rhythm that involves posttranscriptional regulation of microRNA (miRNA) biogenesis by a cycling mRNA-binding protein. In prior work, we observed that a member of the cold-inducible RNA-binding protein family, the RNA-binding motif protein 3 (RBM3) strongly promotes translation. Our preliminary studies now show that manipulation of RBM3 expression has strong and differential effects on miRNA expression that are consistent with effects on the processing of primary and precursor. Indeed, RBM3 associates with and regulates the expression of miRNA processing machinery. miRNAs regulated by RBM3 include those known to regulate synaptic plasticity, neurogenesis and differentiation, neurite out growth, and circadian rhythm. RBM3 expression in euthermic brain is developmentally regulated and is particularly high in regions with high translation rates, especially proliferative zones. Importantly, RBM3 levels fluctuate diurnally under the direct control of cellular clock proteins. We hypothesize that RBM3 regulates miRNA biogenesis and function in a circadian manner in neurons. To test this hypothesis and the impact of this mechanism of translation- dependent processes that are subject to circadian control, we propose four Aims. (1) We will use proteomic, molecular, and biochemical approaches to determine how RBM3 regulates the composition and function of miRNA processing complexes, whether RBM3 binds miRNA precursors as do the mRNA-binding proteins LIN28 and hnRNPA2, and what domains of RBM3 mediate binding. (2) "Antagomirs" and reporter constructs will be used to determine whether regulation of Drosha, Dicer and Ago2 by RBM3 involves direct effects on their translation, or feedback mechanism involving miRNAs that are regulated by RBM3. (3) miRNA array techniques will be used to identify miRNAs under circadian control in brain, and perturbation of RBM3 in synchronized cells will be used to determine which miRNAs cycle because of posttranscriptional regulation by RBM3. Circadian cycling in Drosha and Dicer activity will also be analyzed. (4) Finally, we will address the role of circadian regulation of miRNA expression by RBM3 in processes that are known to be regulated by specific miRNAs and are subject to circadian control: dendritic spine maturation, neurite extension, and differentiation. These studies will describe a novel mechanism for regulating miRNA biogenesis at the pssttranscriptional level that can bias translation to affect critical neuronal events across the circadian cycle. In light of emerging data that disruptions in circadian rhythm and miRNA expression underlie many disease states, our studies should provide important insights into disease-related processes of the nervous system.