Deconvoluting microRNA expression in brain

大脑中 microRNA 表达的去卷积

• 批准号: 8966700
• 负责人: RICHARD H. GOODMAN
• 金额: $ 38.5万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-12-24 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8966700
• 关键词: 3' Untranslated Regions; Affect; Attention; Axon; Biological Assay; Brain; Cell membrane; Cells; Characteristics; Dendrites; Dendritic Spines; Dependence; Elements; Enzymes; Fluorescence; Glutamates; Growth; Growth Cones; Health; Interneurons; Length; Mediating; Membrane; Membrane Protein Traffic; Messenger RNA; Methods; MicroRNAs; Modeling; Monitor; Morphology; Neurons; Pathway interactions; Pattern; Phenotype; Polyribosomes; Porifera; Process; Property; Proteins; Pyramidal Cells; RNA-Binding Proteins; Regulation; Reporter; Signal Transduction; Signaling Molecule; Slice; Somatostatin; Specificity; Stimulus; Synaptic plasticity; Testing; Translations; Vertebral column; axon growth; design; inhibitory neuron; mRNA Transcript Degradation; molecular marker; neuron development; novel; palmitoylation; ratiometric; response; sensor; trafficking

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.

描述（由申请人提供）：在理解 microRNA 对神经元发育的影响方面已经取得了相当大的进展，但 microRNA 也在成熟神经元中表达，并被认为在成熟神经元中控制突触可塑性。这是一个很有吸引力的概念，因为蛋白质翻译的局部调节对于这一过程至关重要，并且 microRNA 可以调节树突生长和脊柱形成，这些特性是可塑性的基础。 microRNA 有助于轴突生长的观点较少受到关注，部分原因是该区室中多核糖体的存在一直存在争议。我们在该提案中认为，microRNA 对轴突生长的调节并不一定需要在轴突内发生翻译，并且由神经元其他地方的 microRNA 调节的棕榈酰化酶可以指导关键信号分子向轴突膜的运输。该提案重点关注 miR-134，这是一种 microRNA，最初以其“活性依赖性”和调节树突棘大小的能力为特征。使用一组比例式 microRNA 传感器，我们意外地发现，成熟皮质神经元中的 miR-134 活性仅限于产生抑制性生长抑素 (SST) 的中间神经元，这与人们广泛持有的 miR-134 功能观点相矛盾。限制 miR-134 表达至 SST 中间神经元的机制尚不清楚，我们认为这是通过 miR-134 前体的细胞特异性加工来实现的。我们将确定神经元产生成熟、功能性 miR-134 的能力是否是由于转录或转录后机制所致，识别与前体相互作用的 RNA 结合蛋白，并使用 miR-Glo（一种新型荧光测定法）测试这些因素是否以细胞特异性方式影响 miR-134 前体的加工。使用一种名为 RISC-trap 的新方法，旨在在 mRNA 降解之前捕获 microRNA-mRNA 相互作用，我们发现 miR-134 靶向棕榈酰化酶 DHHC9，该酶控制 Ras 向细胞膜的运输。我们将测试抑制性 SST 中间神经元中 DHHC9 的 miR-134 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，从而控制 SST 中间神经元中的轴突形态。我们假设 miR-134 的活性调节会对轴突生长产生负面影响，并且与这些细胞独特的轴突分支模式特征有关。 miR-134 等 microRNA 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。