FMRP-regulated association of specific miRNAs with RISC

描述（由申请人提供）：蛋白质表达的时空控制对于神经元发育和可塑性至关重要。脆性X智力低下蛋白（FMRP）是一种RNA结合蛋白，参与神经元中许多特定靶mRNA的调节翻译，尽管FMRP调节翻译的机制尚未明确。RNA诱导的沉默复合物（RISC）也是翻译控制的重要介质，并且最近的证据表明FMRP招募RISC来协同调节编码突触蛋白PSD-95的mRNA的刺激依赖性翻译。然而，FMRP募集RISC的机制在神经元中用于FMRP依赖性翻译调节的程度尚不清楚。基于FMRP广泛调节特定miRNA与RISC复合体的结合以调节突触处的翻译的工作假设，提出以下两个目标：1）鉴定促进重要FMRP靶点GluA 1 mRNA的FMRP依赖性刺激诱导的翻译的特定miRNA;和2）评估Fmr 1-/-改变RISC蛋白Ago 2与突触处特异性miRNA的结合的程度。这些目标将使用原代皮层神经元培养、RNA免疫沉淀、RNA-FISH和免疫荧光显微镜、qRT-PCR和miRNA测序来实现，并且需要神经科学、显微镜和生物信息学方面的培训。这些实验将阐明一种新的机制，FMRP双向调节翻译与精细的空间和时间控制。了解潜在的FMRP依赖的翻译控制机制将提供基本的洞察到正常的大脑发育中涉及的调节蛋白质表达，并阐明神经系统问题，如智力残疾，神经变性，癫痫和自闭症。

DESCRIPTION (provided by applicant): Spatiotemporal control of protein expression is essential for neuronal development and plasticity. Fragile X mental retardation protein (FMRP) is an RNA-binding protein involved in regulated translation of numerous specific target mRNAs in neurons, although the mechanisms by which FMRP regulates translation are not well defined. The RNA-induced silencing complex (RISC) is also an important mediator of translational control and recent evidence indicates that FMRP recruits RISC to cooperatively regulate stimulus-dependent translation of the mRNA encoding the synaptic protein PSD-95. However, it is unknown how widely the mechanism of RISC recruitment by FMRP is used for FMRP-dependent translational regulation in neurons. Based on the working hypothesis that FMRP broadly regulates the association of specific miRNAs with the RISC complex to modulate translation at the synapse, the following two aims are proposed: 1) Identify specific miRNAs that facilitate the FMRP-dependent stimulus-induced translation of an important FMRP target, GluA1 mRNA; and 2) Assess the extent to which Fmr1-/- alters the association of the RISC protein Ago2 with specific miRNAs at the synapse. These aims will be accomplished using primary cortical neuron culture, RNA immunoprecipitation, RNA-FISH and immunofluorescence microscopy, qRT-PCR, and miRNA sequencing, and will require training in neuroscience, microscopy, and bioinformatics. These experiments will illuminate a novel mechanism by which FMRP bi-directionally regulates translation with fine spatial and temporal control. Understanding the mechanisms underlying FMRP-dependent translational control will provide fundamental insight into the regulated protein expression involved in normal brain development and shed light on neurological problems such as intellectual disability, neurodegeneration, epilepsy, and autism.