Understanding the Genome Maintenance Function of the Fragile X Protein (FMRP)

项目总结/摘要 该研究项目的主要目标是了解FMRP的核功能及其在细胞核中的作用。 对脆性X综合征（FXS）病因学基础的影响。当FMR 1基因突变时， 基因导致FMRP功能缺失或丧失。FMRP的主要特点是翻译 在细胞质中广泛的mRNA底物的阻遏物，但它的核功能不是很好 明白我们最近报道，来自FXS患者的细胞遭受全基因组DNA双链损伤， 在复制应力下的断裂（DSB）。此外，FXS细胞中的DNA双链断裂发生在 在基因转录过程中易于形成称为R环的DNA：RNA杂交体的序列。这一发现 提出了FMRP在复制应激期间防止R环诱导的DSB的新功能，从而 维持基因组的稳定性。根据这一范式转变的发现，我们发现FMRP直接结合R- 环和DHX 9，一个R环分解酶，通过多价相互作用。因此，我们的研究提供了 FMRP通过桥接R-环和R-环解析来辅助R-环解析的机制 染色质此外，我们观察到，几乎所有的DNA修复途径的基因表达减少， FXS基因组，有和没有复制应激，并将这种表型与受损的p53途径。在 在这项拟议的研究中，我们将扩展我们的分析，询问FMRP缺乏是否会导致全基因组DNA损伤 在人类神经元中。我们将系统地鉴定和比较诱导的神经元中的DNA双链断裂和R环， 从FXS患者诱导的多能细胞中，通过这样做，我们将辨别出那些最具风险的基因。 在缺乏FMRP的细胞中易受DSB和下调影响，因此提供了一个潜在的 FXS的治疗目标。此外，我们观察到FMRP和DHX 9之间的直接相互作用， RNA：DNA杂合解旋酶。我们将确定FMRP桥接R环和DHX 9的机制 以促进R环分辨率。我们还将探测人类神经元中的FMRP核蛋白质组， 与染色质上的FMRP相互作用的其他因子。我们提出的项目将进一步促进我们的 了解FMRP基因组维持功能，并有望为病因学提供新的线索。 FXS的基础。

PROJECT SUMMARY/ABSTRACT The main goal of the proposed research project is to understand the nuclear functions of FMRP and their impact on the etiological basis for the Fragile X syndrome (FXS). FXS occurs when mutations in the FMR1 gene cause the absence or loss of function of FMRP. FMRP has been primarily characterized as a translation repressor of a wide range of mRNA substrates in the cytoplasm, but its nuclear functions are not well understood. We recently reported that cells derived from FXS patients suffer genome-wide DNA double-strand breaks (DSBs) when under replication stress. Moreover, the DNA DSBs in FXS cells occurred near sequences that are prone to form DNA:RNA hybrids called R-loops during gene transcription. This finding suggested a new function of FMRP in preventing R-loop-induced DSBs during replication stress, thereby maintaining genome stability. Following this paradigm-shifting discovery, we found that FMRP directly binds R- loops and DHX9, an R-loop resolvase, through multivalent interactions. Therefore, our study provides a mechanism through which FMRP assists in R-loop resolution by bridging R-loops and R-loop resolvases on the chromatin. Additionally, we observed reduced gene expression in virtually all DNA repair pathways in the FXS genome, with and without replication stress, and linked this phenotype to an impaired p53 pathway. In this proposed study we will extend our analysis to ask if FMRP deficiency causes genome-wide DNA damage in human neurons. We will systematically identify and compare DNA DSBs and R-loops in neurons induced from FXS patient induced pluripotent cells and by doing so, we will discern those “at-risk” genes that are most susceptible to DSBs and down-regulation in cells lacking FMRP, thus providing a short list of potential therapeutic targets for FXS. In addition, we have observed direct interaction between FMRP and DHX9, an RNA:DNA hybrid helicase. We will determine the mechanism through which FMRP bridges R-loop and DHX9 to facilitate R-loop resolution. We will also probe the FMRP nuclear proteome in human neurons to identify additional factors that interact with FMRP on the chromatin. Our proposed project will further our understanding of the FMRP genome maintenance function and promises to shed new light into the etiological basis for FXS.