Circadian regulation of microRNA biogenesis and function

产品说明：神经元中mRNA翻译的调节是发育和成人大脑过程的关键控制点，这些过程需要基因表达的特定变化。新出现的数据表明，这些过程中的一些-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的机制联系的证据，涉及转录后调节microRNA（miRNA）的生物合成的循环mRNA结合蛋白。在先前的工作中，我们观察到冷诱导RNA结合蛋白家族的成员，RNA结合基序蛋白3（RBM 3）强烈促进翻译。我们的初步研究现在表明，RBM 3表达的操纵对miRNA表达具有强烈和差异性的影响，这与对初级和前体加工的影响一致。事实上，RBM 3与miRNA加工机制相关并调节其表达。由RBM 3调节的miRNA包括已知调节突触可塑性、神经发生和分化、神经突生长和昼夜节律的那些。RBM 3在温脑中的表达是发育调节的，并且在具有高翻译率的区域特别高，特别是增殖区。重要的是，RBM 3水平在细胞时钟蛋白的直接控制下每日波动。我们假设RBM 3在神经元中以昼夜节律的方式调节miRNA的生物发生和功能。为了检验这一假设和这种翻译依赖过程的机制受到昼夜节律控制的影响，我们提出了四个目标。(1)我们将使用蛋白质组学，分子和生物化学方法来确定RBM 3如何调节miRNA加工复合物的组成和功能，RBM 3是否像mRNA结合蛋白LIN 28和hnRNPA 2那样结合miRNA前体，以及RBM 3的哪些结构域介导结合。(2)“拮抗剂”和报告构建体将用于确定RBM 3对Drosha、Dicer和Ago 2的调节是否涉及对其翻译的直接影响，或涉及由RBM 3调节的miRNA的反馈机制。(3)miRNA阵列技术将用于鉴定大脑中昼夜节律控制下的miRNA，并且同步化细胞中RBM 3的扰动将用于确定哪些miRNA由于RBM 3的转录后调节而循环。还将分析Drosha和Dicer活动的昼夜节律循环。(4)最后，我们将讨论RBM 3对miRNA表达的昼夜节律调节在已知由特定miRNA调节并受昼夜节律控制的过程中的作用：树突棘成熟，神经突延伸和分化。这些研究将描述一种在转录水平上调节miRNA生物合成的新机制，该机制可以使翻译偏向于影响昼夜节律周期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表达的破坏是许多疾病状态的基础，我们的研究应该为神经系统疾病相关过程提供重要的见解。 公共卫生相关性：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节步骤。这些过程中的大多数也受昼夜节律的调节，并且在疾病的情况下，当昼夜节律被破坏时，这些过程会受损或被夸大。我们的研究将确定神经元中昼夜节律和蛋白质合成之间的机制联系，为大脑功能和神经系统疾病的控制提供基本见解。

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.