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很重要。FMRP的缺失或极低水平的表达会导致人类脆性X综合征，并在小鼠和果蝇中导致类似的行为和神经解剖缺陷。老鼠和苍蝇的过表达研究也发现了行为和神经解剖学上的缺陷。除了适当的稳态FMRP水平的重要性外，小鼠研究表明，响应突触激活的FMRP水平的瞬态调节对于适当的突触可塑性至关重要。考虑到适当调节FMRP水平的重要性，人们对这种蛋白质的水平是如何调节的知之甚少。最近，我们发现FMRP的苍蝇同源物dFMR1的表达是由siRNA途径的成分调节的。该通路核心成员（AGO2、R2D2和Dicer-2）的缺失会导致dFMR1蛋白在种系和神经系统中的水平显著上调，从而导致dFMR1调控不当导致特定的种系和神经元缺陷。尽管最近的研究已经发现果蝇体内存在一个内源性siRNA通路，但我们的研究结果表明，该通路并没有通过其常规定义的机制来调节dFMR1的表达。事实上，我们已经发现了与其他小RNA途径有关的基因，但在规范的siRNA途径之外，也能调节dFMR1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piRNA途径中的基因，在多个小RNA途径中起作用的基因以及与小RNA途径相互作用以调节dfmr1表达的基因。在第二个主题中，我们将定义由siRNA通路成员调节其所需的dfmr1顺式元件。在第三个子目标中，我们将定义siRNA途径成分调节dfmr1表达的基本机制。这些研究的结果将更精确地定义控制dfmr1表达的新调控途径，为如何调控这一医学上重要的基因提供有价值的信息。公共卫生相关性：对脆性X型智力发育迟滞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型智力发育迟滞、FXTAS和卵巢早衰）至关重要。在本应用中，我们通过实验进一步定义和表征了一种新的调控途径，我们发现该途径对于正确调控dfmr1表达和预防dfmr1错误表达导致的卵巢和神经元缺陷非常重要。这些研究将使我们更好地了解这个医学上重要的基因是如何被调控的，并可能识别出由于FMR1调控不当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.