Circadian regulation of microRNA biogenesis and function

神经元中信使核糖核酸的翻译调控是发育过程的关键控制点 以及需要基因表达特定变化的成人大脑。新出现的数据表明，一些 这些过程--包括突触可塑性、神经发生和记忆形成--也受 昼夜节律。我们已经获得了平移控制和控制之间存在机械联系的证据 昼夜节律，包括通过循环对microRNA(MiRNA)生物发生的转录后调节 MRNA结合蛋白。在之前的工作中，我们观察到冷诱导RNA结合蛋白的一个成员 作为一个家族，RNA结合基序蛋白3(RBM3)强烈促进翻译。我们现在的初步研究 表明操纵RBM3表达对miRNA表达有强烈和不同的影响，这些影响是 与对初级和前驱体加工的影响一致。事实上，RBM3与和 调节miRNA加工机制的表达。受RBM3调控的miRNAs包括已知的 调节突触可塑性、神经发生和分化、轴突生长和昼夜节律。 RBM3在常温脑中的表达受发育调节，在高温度区尤其高 翻译率，特别是增殖区。重要的是，在直接作用下，RBM3水平每天波动 细胞时钟蛋白的控制。我们假设RBM3调控miRNA的生物发生和功能 神经元的昼夜节律。为了检验这一假设和这一翻译机制的影响-- 对于受昼夜节律控制的独立过程，我们提出了四个目标。(1)我们将使用蛋白质组学， 确定RBM3如何调节细胞组成和功能的分子和生化方法 MiRNA加工复合体，RBM3是否像mRNA结合蛋白一样与miRNA前体结合 LIN28和hnRNPA2，以及RBM3的哪些结构域介导结合。(2)“Anagomir”与记者建构 将用于确定RBM3对DROSHA、DICER和Ago2的调节是否涉及对 它们的翻译，或涉及受RBM3调控的miRNAs的反馈机制。(3)miRNA阵列 将使用技术来识别大脑中受昼夜节律控制的miRNAs，以及在大脑中RBM3的扰动 同步的细胞将被用来确定哪个miRNAs周期，因为转录后调控 RBM3.DROSHA和DICER活动的昼夜循环也将被分析。(4)最后，我们将解决角色问题 在已知受特定基因调控的过程中，RBM3对miRNA表达的昼夜调节 MiRNAs和受昼夜节律控制：树突棘成熟、轴突延伸和分化。 这些研究将描述一种在ps转录水平上调节miRNA生物发生的新机制 这可能会使翻译发生偏差，从而影响整个昼夜节律中的关键神经元事件。根据新出现的数据 昼夜节律和miRNA表达的紊乱是许多疾病状态的基础，我们的研究应该 提供对神经系统疾病相关过程的重要见解。

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.