Deconvoluting microRNA expression in brain

描述(由申请人提供)：在了解microRNAs对神经元发育的影响方面已经取得了相当大的进展，但microRNAs也在成熟神经元中表达，在那里它们被认为是控制突触可塑性的。这是一个有吸引力的概念，因为蛋白质翻译的局部调节在这一过程中是必不可少的，而microRNAs可以调节树突生长和棘突的形成，这是可塑性的基础。关于microRNAs有助于轴突生长的观点受到的关注较少，部分原因是这个隔室中是否存在多聚核糖体一直存在争议。在这一建议中，我们认为microRNA对轴突生长的调节不一定需要在轴突内发生翻译，棕榈酰化酶受神经元其他地方的microRNA调控，可以将关键的信号分子运输到轴突膜上。这项提议的重点是miR-134，这是一种微小RNA，最初的特征是它的“活性依赖”和调节树突棘大小的能力。使用一组比率计量型microRNA传感器，我们出人意料地发现，成熟皮质神经元中的miR-134活性仅限于产生生长抑素(SST)的中间神经元，这与人们普遍认为的miR-134功能的观点相矛盾。将miR-134限制在SST-中间神经元表达的机制尚不清楚，我们认为这是通过对miR-134前体进行细胞特异性处理来实现的。我们将建立神经元产生成熟、有功能的miR-134的能力是由于转录还是转录后机制，识别与前体相互作用的RNA结合蛋白，并使用一种新的荧光分析方法miR-Glo测试这些因素是否影响以细胞特有的方式处理miR-134前体。使用一种名为RISC-TRAP的新方法，我们发现miR-134针对棕榈酰化酶DHHC9，该酶控制RAS向细胞膜的运输。我们将测试miR-134对SST抑制中间神经元中DHHC9的调节，以及随后RAS的棕榈酰化是否控制RAS运输到轴突生长锥体，从而控制SST中间神经元中的轴突形态。我们假设miR-134的活性调节对轴突生长有负面影响，并与这些细胞独特的轴突分支模式特征有关。MiR-134等microRNAs调节棕榈酰化酶的能力，从而调节RAS等信号分子的膜运输，可能是突触可塑性的重要组成部分，特别是与轴突生长有关。

DESCRIPTION (provided by applicant): Considerable progress has been made understanding the effects of microRNAs on neuronal development, but microRNAs are also expressed in mature neurons where they have been proposed to control synaptic plasticity. This is an appealing concept because local regulation of protein translation is essential for this process and microRNAs can regulate dendritic growth and spine formation, properties that underlie plasticity. The idea that microRNAs contribute to axonal growth has received less attention, in part because the existence of polyribosomes in this compartment has been controversial. We argue in this proposal that microRNA regulation of axonal growth does not necessarily require translation to occur within the axon and that palmitoylation enzymes, regulated by microRNAs elsewhere in the neuron, can direct trafficking of key signaling molecules to axonal membranes. This proposal focuses on miR-134, a microRNA initially characterized by virtue of its "activity-dependence" and ability to regulate dendritic spine size. Using a set of ratiometric microRNA sensors, we found, unexpectedly, that miR-134 activity in mature cortical neurons was limited to inhibitory somatostatin (SST)-producing interneurons, contradicting a widely held view of miR-134 function. The mechanisms responsible for restricting miR-134 expression to SST-interneurons are unknown, and we propose that this is accomplished via cell-specific processing of the miR-134 precursor. We will establish whether the ability of neurons to generate mature, functional miR-134 is due to transcriptional or post-transcriptional mechanisms, identify RNA-binding proteins that interact with the precursor, and test whether these factors affect processing of the miR-134 precursor in a cell- specific manner using miR-Glo, a novel fluorescence assay. Using a new method termed RISC-trap designed to capture microRNA-mRNA interactions prior to mRNA degradation, we discovered that miR-134 targets the palmitoylation enzyme, DHHC9, which controls Ras trafficking to the cell membrane. We will test whether the miR-134 regulation of DHHC9 in inhibitory SST interneurons, and the consequent palmitoylation of Ras, controls Ras trafficking to axonal growth cones and, consequently, axon morphology in SST interneurons. We hypothesize that activity-regulation of miR- 134 negatively influences axon growth and is related to the unique axonal branching pattern characteristic of these cells. The ability of microRNAs such as miR-134 to regulate palmitoylation enzymes, and thereby membrane trafficking of signaling molecules like Ras, could be an important component of synaptic plasticity, particularly in relation to axonal growth.