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的脆性X智力低下蛋白1(FMRP)的研究表明，它的适当表达对正常的发育和行为非常重要。FMRP的缺失或非常低的表达水平会导致人类的脆性X综合征，以及小鼠和果蝇的类似行为和神经解剖学缺陷。在老鼠和苍蝇身上的过度表达研究也发现了行为和神经解剖学上的缺陷。除了适当的稳定水平的FMRP的重要性外，在小鼠的研究表明，突触激活时FMRP水平的瞬时调节对于正常的突触可塑性至关重要。鉴于适当调节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的表达受siRNA途径的成分调控。失去这一途径的核心成员(AGO2、R2D2和DICER-2)会导致生殖系和神经系统中dFMR1蛋白水平的显著上调，从而导致特定的生殖系和神经元缺陷，这是由于dFMR1调控不当所致。虽然最近的研究已经证实在果蝇中存在一个功能正常的内源性siRNA途径，但我们的结果表明，这一途径并不是通过其规范定义的机制来调节dFMR1的表达。事实上，我们已经发现了参与其他小RNA途径的基因，但在规范的siRNA途径之外，也调节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，作用于多个小RNA途径的基因，以及与小RNA途径相互作用的基因，以调节dfmr1的表达。在第二个子目标中，我们将定义siRNA途径成员调控其所需的dfmr1顺式元件。在第三个子目标中，我们将定义siRNA途径组件调节dfmr1表达的基本机制。这些研究的结果将更准确地定义一种控制dfmr1表达的新的调控途径，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)来说，对脆性X智力低下的适当调控是至关重要的。在这一应用中，我们提出了进一步定义和表征一种新的调控途径的实验，我们发现这一调控途径对于正确调控dfmr1的表达和预防由于dfmr1错误表达而导致的卵巢和神经元缺陷非常重要。这些研究将更好地了解这种医学上重要的基因是如何调控的，并可能识别由于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.