Regulation of the Drosophila Fragile X Protein by siRNA Pathway Components

描述（由申请人提供）：对人类及其小鼠和苍蝇同源物中脆性X智力迟钝蛋白1（FMRP）的研究表明，其正确表达对正常发育和行为至关重要。 FMRP表达的缺失或非常低的水平导致人类的脆性X综合征以及小鼠和苍蝇的类似行为和神经解剖缺陷。 在小鼠和苍蝇中的过度表达研究也发现了行为和神经解剖缺陷。除了FMRP的适当稳态水平的重要性之外，在小鼠中的研究已经表明，响应于突触激活的FMRP水平的瞬时调节对于适当的突触可塑性是至关重要的。 鉴于FMRP水平的适当调节的重要性，很少有人知道如何调节这种蛋白质的水平。最近，我们发现FMRP的果蝇同源物（称为dFMR 1）的表达受siRNA途径组分的调节。 该途径的核心成员（AGO 2、R2D2和Dicer-2）的缺失导致种系和神经系统中dFMR 1蛋白水平的显著上调，这导致由于dFMR 1失调而导致的特定种系和神经元缺陷。虽然最近的研究已经确定，有一个功能性的内源性siRNA途径在苍蝇，我们的研究结果表明，这条途径并没有被用来调节dFMR 1的表达，通过其规范定义的机制。 事实上，我们已经发现了参与其他小RNA途径的基因，但在典型的siRNA途径之外，它们也调节dFMR 1的表达。在第一个子目标中，我们将筛选一组候选基因，其中包括皮尔纳途径中的基因、在多个小RNA途径中发挥作用的基因以及与小RNA途径相互作用以调节dfmr 1表达的基因。在第二个子目标中，我们将定义dfmr 1顺式元件所需的siRNA途径成员的调控。 在第三个子目标中，我们将定义siRNA途径组分调节dfmr 1表达的基本机制。这些研究的结果将更精确地定义一个新的调控途径，控制dfmr 1的表达，提供有价值的信息，如何在医学上重要的基因是调节。公共卫生相关性：脆性X智力低下的适当调节对于正常认知和预防至少三种人类疾病（脆性X智力低下、FXTAS和卵巢早衰）至关重要。在本申请中，我们提出了进一步定义和表征一种新的调节途径的实验，我们发现该途径对于正确调节dfmr 1表达和预防由于dfmr 1错误表达引起的卵巢和神经元缺陷是重要的。这些研究将更好地了解这种医学上重要的基因是如何调节的，并可能识别由于FMR 1失调而导致其他人类疾病的其他基因。

DESCRIPTION (provided by applicant): Studies of the Fragile X Mental Retardation Protein 1 (FMRP) in humans and its mouse and fly homologs have shown that its proper expression is important for proper development and behavior. Loss, or very low levels of FMRP expression, leads to Fragile X syndrome in humans and analogous behavioral and neuro-anatomical defects in mice and flies. Over-expression studies in the mouse and fly have identified behavioral and neuro-anatomical defects as well. In addition to the importance of proper steady state levels of FMRP, studies in the mice have shown that transient modulation of FMRP levels in response to synaptic activation are crucial for proper synaptic plasticity. Given the importance of the proper regulation of FMRP levels, very little is known about how the levels of this protein are regulated. Recently we discovered that expression of the fly homologue of FMRP, called dFMR1, is regulated by components of the siRNA pathway. Loss of the core members of this pathway (AGO2, R2D2 and Dicer-2) leads to significant upregulation of dFMR1 protein levels in the germline and nervous system that cause specific germ line and neuronal defects due to dFMR1 misregulation. Although recent studies have identified that there is a functioning endogenous siRNA pathway in flies, our results indicate that this pathway is not being used to regulate dFMR1 expression through its canonically defined mechanism. In fact we have found genes, involved in other small RNA pathways, but outside of the canonical siRNA pathway, that also regulate dFMR1 expression. In the first subaim we will screen a candidate set of genes that includes genes in the piRNA pathway, genes that act in multiple small RNA pathways as well as genes that interact with small RNA pathways for a role in regulating dfmr1 expression. In the second subaim we will define the dfmr1 cis-elements required for its regulation by the siRNA pathway members. In the third subaim we will define the basic mechanism by which the siRNA pathway components regulate dfmr1 expression. Results from these studies will more precisely define a novel regulatory pathway that controls dfmr1 expression, providing valuable information as to how this medically important gene is regulated. PUBLIC HEALTH RELEVANCE: The proper regulation of the Fragile X Mental Retardation is fundamentally important for normal cognition and the prevention of at least three human diseases (Fragile X mental retardation, FXTAS and premature ovarian failure). In this application we put forth experiments to further define and characterize a novel regulatory pathway that we have found to be important for the proper regulation dfmr1 expression and the prevention of ovarian and neuronal defects due to dfmr1 misexpression. These studies will provide a better understanding about how this medically important gene is regulated, as well as may identify other genes that lead to other human diseases due to FMR1 misregulation.