Unraveling how RNA helicase Mov10 regulates Ago2 function

人类和动物的大脑由数十亿个被称为神经元的特殊细胞组成，这些细胞共同行动，通过我们不完全了解的机制来驱动行为、学习和记忆。神经元之间通过其长的细胞延伸部分相互通信，这些细胞延伸部分由细胞中信使RNA产生的蛋白质组成。这项研究的主题是一种最近表征的因子Mov10，它通过解开信使RNA来调节蛋白质的产生，以促进它们转化为蛋白质。Mov10对于跨物种的早期胚胎发育和正常的神经元发育是必要的，包括产生连接神经元的分支。这项工作的目标是识别Mov10解离的RNA，了解Mov10？S解离活动是如何调控的，并确定Mov10如何参与对神经元功能至关重要的分支的产生。该项目将包括对高中生、本科生和研究生进行科学方法论和科学过程的培训，包括假设检验。此外，这项工作将为PI的公开演讲提供信息，向地方和国家专业团体介绍科学发现和支持基础研究的重要性。蛋白质的突然翻译是正常发育和神经元功能所必需的，但尚不清楚这一过程是如何调节的。本研究的目的是探索RNA解旋对RNA命运的关键作用。RNA解旋酶Mov10与3‘端非编码区中的microRNA识别元件(MRE)结合。它解开RNA中的二级结构，使miRNA途径的主要效应蛋白ArgAerte2能够与MRE结合。在发育中的脑中，Mov10与编码神经元投射和细胞骨架蛋白的mRNAs结合，提示Mov10在调节树突的翻译中起作用。正在检验的假设是，Mov10与稳定的RNA二级结构RNAG-四链(GQs)结合，以调节Ago2与3UTR中MRE的关联。将使用大脑特异的Mov10条件基因敲除小鼠(CKO)识别依赖于Mov10的Ago2靶向mRNAs，在CKO上将执行Ago2增强剪辑(ECLIP)。稳定表达是Mov10依赖的RNA将通过RNA-seq和基因本体学来鉴定，以鉴定WT和Mov10 CKO脑中的细胞骨架RNA。靶mRNAs中的MRE将通过突变报告结构中表达的3‘UTRs来鉴定。对Mov10活性的调节将通过检验Mov10的磷酸化控制其解旋酶活性的假设来检验。模型GQ，iSpinach，将被用来研究Mov10磷酸化模拟物是如何解开GQ的。还将研究Mov10如何通过扩展Mov10杂合性丢失导致培养神经元树突状突起减少的观察结果来调节神经元树突的形成。正在测试的假设是，Mov10调节Ago2与神经元投射和细胞骨架mRNAs的联系。Mov10/Ago2调控的细胞骨架RNA将通过将Ago2调控区域已被移除的候选基因引入Mov10 CKO神经元来识别。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Human and animal brains are made up of billions of specialized cells called neurons that act together to drive behavior, learning and memory through the mechanisms that we do not fully understand. Neurons communicate with each other through their long cellular extensions that are composed of proteins, produced in the cells from messenger RNAs. The subject of this research is a recently characterized factor, Mov10, that regulates protein production by unwinding messenger RNAs to facilitate their translation into proteins. Mov10 is necessary for early embryonic development across species and for normal neuron development that includes producing the branches that connect neurons. The goals of this work are to identify the RNAs that Mov10 unwinds, to understand how Mov10?s unwinding activity is regulated and to determine how Mov10 participates in the production of the branching that is critical for neuronal function. This project will involve the training of high school students, undergraduates and graduate students in both scientific methodology and in the scientific process, including hypothesis testing. In addition, this work will inform public talks by the PI for presentation to local and state professional groups about scientific discoveries and the importance of supporting basic research.Bursts of protein translation are required for normal development and neuronal function but it is unknown how this process is regulated. The goal of this research is to explore the key role of RNA unwinding on RNA fate. RNA helicase Mov10 binds in proximity to MicroRNA Recognition Elements (MREs) in 3'UTRs. It unwinds secondary structures in RNA to allow access of the primary effector protein of the miRNA pathway, Argonaute 2, to MREs. Mov10 was shown to bind mRNAs encoding neuronal projection and cytoskeletal proteins in developing brain, suggesting a role for Mov10 in regulating translation in dendrites. The hypothesis being tested is that Mov10 binds RNA G-quadruplexes (GQs), which are stable RNA secondary structures, to modulate Ago2 association to MREs in the 3?UTR. The Mov10-dependent Ago2 target mRNAs will be identified using a brain-specific Mov10 conditional knockout mouse (cko) on which Ago2-enhanced CLIP (eCLIP) will be performed. RNAs whose steady-state expression is Mov10-dependent will be identified by RNA-seq followed by Gene Ontology to identify cytoskeletal RNAs in both WT and Mov10 cko brain. MREs in the target mRNAs will be identified by mutagenesis of the 3'UTRs expressed in a reporter construct. Regulation of Mov10 activity will be examined by testing the hypothesis that phosphorylation of Mov10 controls its helicase activity. The model GQ, iSpinach, will be used to examine how Mov10 phospho-mimics that are either constitutively phosphorylated or constitutively unphosphorylated unwind GQs. It will also be studied how Mov10 regulates neuronal dendrite formation by expanding on the observation that heterozygous loss of Mov10 leads to reduced dendritic arbors in cultured neurons. The hypothesis being tested is that Mov10 regulates Ago2 association with neuron projection and cytoskeleton mRNAs. The Mov10/Ago2-regulated cytoskeletal RNAs that mediate arborization will be identified by introducing candidate genes from which the Ago2-regulatory regions have been removed into Mov10 cko neurons.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.