Investigation of Fragile X mental retardation protein interactions with the miRNA pathway.

项目摘要 脆性X智力低下综合征是遗传性智力低下最常见的形式， 男性3600例，女性约6000例。该综合征是由正常细胞蛋白质的丢失引起的， 将其命名为脆性X智力低下蛋白（FMRP）。尽管在过去的二十年里进行了广泛的研究， FMRP缺失与脆性X综合征表型之间的关系尚不完全 明白FMRP是一种RNA结合蛋白，参与特异性RNA的转运和翻译调节。 信使RNA（mRNA）靶点。生物化学研究已经确定FMRP使用其甘氨酸-甘氨酸- 甘氨酸（RGG）盒以高亲和力结合形成G四链体（GQ）结构的RNA序列。的 FMRP发挥其翻译调节剂功能的机制尚不清楚，然而， 提出，该蛋白质与microRNA（miRNA）通路一起调节局部蛋白质 对突触输入作出反应的合成。这项建议的具体目标如下： 1.研究FMRP在miRNA成熟中的作用。一些miRNAs已经被证明需要 FMRP发挥其功能，并且FMRP也已被证明结合pre-miRNA。我们假设 FMRP通过与由其形成的GQ结构的相互作用来调节特定miRNA的成熟。 pre-miRNAs前体。为了验证这一假设，我们将描述预测在2000年形成的GQ结构。 选择pre-miRNAs并分析它们与FMRP的相互作用。随后，我们将分析 FMRP对Dicer产生成熟miRNA的影响。 2. FMRP：GQ mRNA：miRNA相互作用在翻译调控中的研究FMRP显示 通过与miRNA引导的RNA相互作用直接调节特定mRNA的翻译 诱导沉默复合物（RISC）。遵循FMRP与GQ结构相互作用的指导假设， 特异性mRNA来调节它们被RISC识别，在此目标下，我们将分析FMRP：GQ mRNA： 在具有预测的GQ结构的其他mRNA的背景下，miRNA相互作用：（i）在它们的miRNA内， 结合位点和（ii）紧邻它们。 3.研究各种FMRP亚型与miRNA途径的相互作用。我们假设 在外显子12处通过选择性剪接产生的FMRP异构体将与miRNA具有不同的相互作用 前体和/或蛋白质组分的miRNA途径，这可能会导致功能性 在翻译规则方面的差异。此外，我们假设FMRP活性依赖性 初级和次级磷酸化事件可进一步调节这些功能差异。

Project Summary Fragile X mental retardation syndrome is the most common form of inherited mental retardation, affecting ~ 1 in 3600 males and ~ 1 in 6000 females. The syndrome is caused by the loss of a normal cellular protein, named the fragile X mental retardation protein (FMRP). Despite extensive research in the past two decades, the relationship between the absence of FMRP and the phenotype of the fragile X syndrome is still not fully understood. FMRP is an RNA binding protein involved in the transport and translation regulation of specific messenger RNA (mRNA) targets. Biochemical studies have determined that FMRP uses its arginine-glycine- glycine (RGG) box to bind with high affinity to RNA sequences that form G quadruplex (GQ) structures. The mechanisms by which FMRP exerts its translation regulator function are not known, however it has been proposed that the protein works in conjunction with the microRNA (miRNA) pathway to regulate local protein synthesis in response to synaptic input. This proposal has the following specific aims: 1. Investigation of the FMRP role in miRNA maturation. Several miRNAs have been shown to require FMRP to exert their function, and FMRP has also been shown to bind pre-miRNAs. We hypothesize that FMRP regulates the maturation of specific miRNAs through its interactions with GQ structures formed by their precursor pre-miRNAs. To test this hypothesis we will characterize the GQ structures predicted to form in selected pre-miRNAs and analyze their interactions with FMRP. Subsequently, we will analyze the effect of FMRP upon the production of mature miRNAs by Dicer. 2. Investigation of FMRP: GQ mRNA: miRNA interactions in translation regulation. FMRP has been shown to directly regulate the translation of specific mRNAs through its interactions with the miRNA-guided RNA induced silencing complex (RISC). Following the guiding hypothesis that FMRP interacts with GQ structures in specific mRNAs to modulate their recognition by RISC, under this aim we will analyze FMRP: GQ mRNA: miRNA interactions in the context of other mRNAs that have predicted GQ structures: (i) within their miRNA binding sites and (ii) immediately adjacent to them. 3. Investigation of various FMRP isoforms interactions with the miRNA pathway. We hypothesize that the FMRP isoforms created by alternative splicing at exon 12 will have different interactions with miRNA precursors and/or protein components of the miRNA pathway, which could potentially result in functional differences with respect to translation regulation. Additionally, we hypothesize that FMRP activity-dependent primary and secondary phosphorylation events could further modulate these functional differences.