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

项目摘要/摘要 拟议研究项目的主要目标是了解FMRP的核功能及其 对脆性X综合征(FXS)病因基础的影响。当FMR1突变时会发生FXS 基因导致FMRP功能缺失或丧失。FMRP的主要特征是翻译 在细胞质中有广泛的mRNA底物抑制物，但其核功能不佳 明白了。我们最近报道了来自FXS患者的细胞遭受全基因组DNA双链 在复制压力下中断(DSB)。此外，FXS细胞中的DNA双链断裂发生在 容易形成DNA的序列：在基因转录过程中被称为R-环的RNA杂交体。这一发现 提出了FMRP在防止复制应激时R环诱导的DSB中的新功能，从而 保持基因组的稳定。根据这一范式转换的发现，我们发现FMRP直接结合R- 环和DHX9，一种R-环解析酶，通过多价相互作用。因此，我们的研究提供了一个 FMRP通过桥接R-循环和R-循环解析来帮助R-循环解析的机制 染色质。此外，我们观察到几乎所有DNA修复途径中的基因表达都减少了 FXS基因组，在有和没有复制压力的情况下，并将这种表型与受损的p53途径联系起来。在……里面 在这项拟议的研究中，我们将扩展我们的分析，以询问FMRP缺陷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DNADSB和R环在诱导的神经元中的作用 从FXS患者诱导的多能细胞中，通过这样做，我们将辨别出最危险的基因 对DSB敏感，在缺乏FMRP的细胞中下调，从而提供了一个简短的潜在列表 FXS的治疗靶点。此外，我们还观察到FMRP和DHX9之间的直接相互作用，以及 RNA：DNA杂合解旋酶。我们将确定FMRP连接R-loop和DHX9的机制 以促进R-环路分辨率。我们还将探索人类神经元中的FMRP核蛋白质组，以识别 与染色质上的FMRP相互作用的其他因素。我们提议的项目将进一步推动我们的 对FMRP基因组维持功能的理解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.