Identification and Validation of FMRP Target RNAs

描述(由申请人提供)：这是一项建议，旨在测试FMRP未能结合特定RNA导致脆性X智力低下的假设，并确定这些RNA。这一建议的基础是我们发现了FMRP RGG盒和Kh2结构域的序列和结构特异性靶点，后者与1例在该RNA结合域存在错义突变(I304N)的患者的严重疾病有关。我们通过证明它与脑多聚核糖体的FMRP竞争，将Kh2 RNA靶标，称为接吻复合体RNA，与疾病联系在一起，这表明FMRP利用这个RNA基序来影响特定mRNAs的翻译调节。这里将明确讨论FMRP序列特异性RNA结合的三个方面。首先，将使用几种方法识别FMRP RNA靶标：辅之以结构研究的生物信息筛选，免疫共沉淀RNA的微阵列分析，在没有FMRP的情况下其在多聚核糖体上的分布发生变化的RNA的微阵列分析，以及交联IP(CLIP)研究。其次，为了帮助在体内验证这些新的RNA靶点，我们将产生三个新的脆性X智力低下小鼠模型。第三，这些新的模型系统将与现有的FMR1缺失小鼠结合使用，以验证小鼠大脑中已识别的FMRP的RNA靶标。综上所述，这些研究将解决这样的假设，即与I304N突变相关的智力低下，以及更广泛的脆性X综合征，可能与含有接吻复杂基序的RNA作为FMRP翻译调控目标的关键作用有关。

DESCRIPTION (provided by applicant): This is a proposal to test the hypothesis that Fragile-X mental retardation results from a failure of FMRP to bind specific RNAs, and to identify those RNAs. The foundation of this proposal is our finding of sequence and structure-specific targets for the FMRP RGG box and KH2 domain, the latter of which is associated with severe disease in a patient with a missense mutation (I304N) in this RNA-binding domain. We have related the KH2 RNA target, termed a kissing complex RNA, to disease by demonstrating that it competes FMRP off of brain polyribosomes, suggesting that this RNA motif is used by FMRP to effect translational regulation of specific mRNAs. Three aspects of FMRP sequence-specific RNA binding will be definitively addressed here. First, FMRP RNA targets will be identified using several methods: bioinformatic screens complemented by structural studies, microarray analysis of coimmunoprecipitating RNAs, microarray analysis of RNAs whose distribution on polyribosomes is shifted in the absence of FMRP, and cross-linking IP (CLIP) studies. Second, to help validate these new RNA targets in vivo, we will generate three new mouse models of Fragile-X mental retardation. Third, these new model systems will be used in conjunction with the existing FMR1-null mouse to validate identified RNA targets of FMRP in mouse brain. Taken together, these studies will address the hypothesis that mental retardation associated with the I304N mutation, and likely the Fragile-X syndrome more generally, may relate to a crucial role for RNAs harboring the kissing complex motif as targets for FMRP translational regulation.