Circadian regulation of microRNA biogenesis and function

描述：神经元中信使核糖核酸翻译的调节是控制发育和成人大脑过程的一个关键点，这些过程需要基因表达的特定变化。新出现的数据表明，许多这样的过程--包括突触可塑性、神经发生和记忆形成--也受到昼夜节律的调节。我们已经获得了翻译控制和昼夜节律之间存在机械联系的证据，这种联系涉及到一个循环的mRNA结合蛋白对microRNA(MiRNA)生物发生的转录后调节。在以前的工作中，我们观察到作为冷诱导RNA结合蛋白家族的成员，RNA结合基序蛋白3(RBM3)强烈促进翻译。我们的初步研究表明，操纵RBM3的表达对miRNA的表达具有强烈的差异性影响，这与对初级和前体加工的影响是一致的。事实上，RBM3与miRNA加工机制有关并调节其表达。受RBM3调控的miRNAs包括那些已知调节突触可塑性、神经发生和分化、轴突生长和昼夜节律的miRNAs。RBM3在亚温脑中的表达受发育调节，在高翻译率的区域尤其是增殖区表达特别高。重要的是，在细胞时钟蛋白的直接控制下，RBM3的水平每天都会波动。我们假设RBM3以昼夜节律的方式调节神经元中miRNA的生物发生和功能。为了检验这一假设以及这一受昼夜节律控制的翻译依赖过程的机制的影响，我们提出了四个目标。(1)我们将使用蛋白质组学、分子和生化方法来确定RBM3如何调节miRNA加工复合体的组成和功能，RBM3是否像mRNA结合蛋白LIN28和hnRNPA2一样与miRNA前体结合，以及RBM3的哪些结构域介导结合。(2)“对应物”和报告结构将用于确定RBM3对DROSHA、DICER和Ago2的调控是否涉及对其翻译的直接影响，或涉及受RBM3调控的miRNA的反馈机制。(3)将使用miRNA阵列技术来识别大脑中受昼夜节律控制的miRNAs，并将利用同步细胞中RBM3的扰动来确定由于RBM3的转录后调控而导致哪个miRNAs周期。DROSHA和DICER活动的昼夜循环也将被分析。(4)最后，我们将讨论RBM3对miRNA表达的昼夜调控在已知受特定miRNAs调控并受昼夜节律控制的过程中的作用：树突棘成熟、轴突延伸和分化。这些研究将描述一种在转录水平上调节miRNA生物发生的新机制，该机制可以使翻译产生偏差，从而影响整个昼夜节律中的关键神经元事件。鉴于新出现的数据表明，昼夜节律和miRNA表达的紊乱是许多疾病状态的基础，我们的研究应该为神经系统的疾病相关过程提供重要的见解。与公共健康相关：神经元中新蛋白质的合成代表了与大脑发育、记忆形成和疾病相关的广泛过程中的关键调控步骤。这些过程中的大多数也受到昼夜节律的调节，当昼夜节律被打乱时，这些过程会受到损害，或者在疾病的情况下被夸大。我们的研究将确定昼夜节律和神经元中蛋白质合成之间的机械联系，为控制大脑功能和神经疾病提供基本的见解。

DESCRIPTION: 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 transcriptional 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. PUBLIC HEALTH RELEVANCE: Synthesis of new proteins in neurons represents a critical regulatory step in a wide range of processes related to brain development, memory formation, and disease. Most of these processes are also regulated by circadian rhythm and are impaired, or exaggerated in the case of disease, when circadian rhythms are disrupted. Our studies will define a mechanistic link between circadian rhythm and protein synthesis in neurons, providing fundamental insights into the control of brain function and neurological diseases.