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Mechanism and Function Of MBNL Mediated mRNA Localization in Neuronal Development and Neurologic Disease

MBNL介导的mRNA定位在神经元发育和神经系统疾病中的机制和功能

基本信息

批准号：
10553695
负责人：
GARY J BASSELL
金额：
$ 41.2万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-01 至 2025-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10553695
关键词：
Adult Alternative Splicing Axon Basic Science Biochemical Brain Diseases CUG repeat Cell Nucleus Cell physiology Cells Cellular biology Charcot-Marie-Tooth Disease Complex Cytoplasm Cytoplasmic Granules Cytoskeleton Dendrites Destinations Development Disease Distal Elements Exhibits FMRP Fragile X Syndrome Genomics Hippocampus Impairment In Vitro Investigation Kinesin Kinetics Link Maintenance Mediating Membrane Messenger RNA Microtubules Mitochondria Modeling Morphology Motor Mus Muscle Mutate Myotonic Dystrophy Nerve Degeneration Neurites Neurologic Neurons Neuropil Pathogenesis Patients Process Property Proteins RNA RNA Splicing RNA Transport RNA-Binding Proteins Records Regulation Reporting Research Resolution Signal Transduction Site Skeletal Muscle Symptoms Synapses Synaptic Vesicles Synaptic plasticity Translating Translations Variant biophysical properties cell motility live cell imaging mouse model nervous system disorder neuron development novel postsynaptic proteostasis synaptic function vesicular release

项目摘要

The development and maintenance of neurons and synaptic connections are highly complex processes, in part due to the massive cytoplasmic volume and complex branching morphology of axons and dendrites. As one mechanism, it is well appreciated that RNA localization and local translation are required to precisely regulate protein homeostasis at synapses. Indeed, loss of FMRP in Fragile X Syndrome, or other impairments to RNA localization and local translation at synapses, likely contribute to brain disorders. To better understand RNA localization and local translation in neurons, we must elucidate the RNA cis-elements, RBP trans-factors, and cytoskeletal motors mediating these processes. Although ongoing efforts have demonstrated how RNA binding proteins (RBPs) can regulate local translation at post-synaptic sites, there still exists a major gap in our understanding of how RBPs transport RNAs to regulate synaptic function. Fortunately, recent observations provide clues about fruitful lines of investigation. For example, multiple studies report that distally localized RNAs are enriched for cis-elements targeted by Muscleblind-like (MBNL) proteins. Although these observations suggest that MBNL may be a major player in localizing RNAs to the pre- and post-synapse, we still lack a mechanistic understanding for how MBNL proteins may achieve this task, or what functions depend on MBNL-mediated RNA localization. This line of research has important implications for the neurological disease myotonic dystrophy (dystrophia myotonica, DM), in which MBNLs are depleted by toxic CUG repeats. Therefore, an emerging hypothesis is that RNA localization functions of MBNL are important for proper synapse function, and that mis-localized RNAs might account for some neurological features of DM patients, particularly early in disease. Here, using MBNL depletion and DM-associated models, we propose to identify specific functions for the localization of MBNL targets. Aim 1 will elucidate mechanisms of MBNL-mediated mRNA localization in neurons. We will define the RNA targets that are localized by MBNL in the pre- and post-synapse. We will characterize dynamic properties of motile MBNL RNA granules in live neurons and identify cytoskeletal motors and adaptors associated with these granules. Using genomics, live cell imaging, and biochemical approaches, we will establish mechanisms of how MBNL-interacting RNAs are transported. Aim 2 will define functions conferred by MBNL-dependent RNA localization using models of synapse development and function, and models of myotonic dystrophy. By depleting cytoplasmic MBNL and other proteins required for MBNL-dependent RNA localization, we will assess cellular functions dependent on this process. We will identify specific neuronal functions, such as synaptic vesicle release, that depend on proper localization of mRNAs by MBNL proteins. The impact of this research is to better understand how RNA localization and local translation confers important synaptic functions and how they may go awry in DM. As few RNA binding proteins have been linked to motors, this may evolve into a unifying model for mRNA transport to synapses.

神经元和突触连接的发育和维持是高度复杂的过程，部分地由于大量的细胞质体积和轴突和树突的复杂分支形态。作为一个机制，众所周知，需要RNA定位和局部翻译来精确调节突触的蛋白质稳态。事实上，脆性 X 综合征中 FMRP 的缺失，或 RNA 的其他损伤突触的定位和局部翻译可能会导致大脑疾病。为了更好地理解RNA 为了实现神经元的定位和局部翻译，我们必须阐明 RNA 顺式元件、RBP 反式元件和细胞骨架马达介导这些过程。尽管持续的努力已经证明了RNA如何结合蛋白质（RBP）可以调节突触后位点的局部翻译，但我们的研究仍存在重大差距了解 RBP 如何转运 RNA 来调节突触功能。幸运的是，最近的观察提供有关卓有成效的调查路线的线索。例如，多项研究报告称，远端局部 RNA 富含肌盲样 (MBNL) 蛋白靶向的顺式元件。虽然这些观察表明 MBNL 可能是 RNA 定位到突触前和突触后的主要参与者，我们对于 MBNL 蛋白如何完成这项任务，或者依赖什么功能仍然缺乏机制理解 MBNL介导的RNA定位。该研究对神经学具有重要意义疾病性强直性肌营养不良（肌强直性营养不良，DM），其中 MBNL 被有毒的 CUG 重复耗尽。因此，一个新的假设是 MBNL 的 RNA 定位功能对于正确的定位非常重要。突触功能，以及错误定位的 RNA 可能解释了 DM 患者的一些神经学特征，特别是在疾病早期。在这里，我们建议使用 MBNL 耗尽和 DM 相关模型来识别 MBNL 目标定位的特定功能。目标 1 将阐明 MBNL 介导的机制 mRNA 在神经元中的定位。我们将定义 MBNL 在突触前和突触后定位的 RNA 靶标。我们将表征活神经元中运动 MBNL RNA 颗粒的动态特性，并鉴定与这些颗粒相关的细胞骨架马达和适配器。使用基因组学、活细胞成像和通过生化方法，我们将建立 MBNL 相互作用 RNA 的运输机制。目标2 将使用突触发育模型定义 MBNL 依赖性 RNA 定位所赋予的功能强直性肌营养不良的功能和模型。通过消耗细胞质 MBNL 和其他所需蛋白质对于 MBNL 依赖性 RNA 定位，我们将评估依赖于此过程的细胞功能。我们将识别特定的神经元功能，例如突触小泡释放，这取决于神经元的正确定位 MBNL 蛋白的 mRNA。这项研究的影响是更好地了解 RNA 定位和局部翻译赋予重要的突触功能以及它们如何在 DM 中出错。尽可能少的RNA结合蛋白质已与马达相连，这可能会演变成 mRNA 运输到突触的统一模型。