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

了解脆性 X 蛋白 (FMRP) 的基因组维持功能

• 批准号: 10511129
• 负责人: WENYI FENG
• 金额: $ 24.45万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-15 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10511129
• 关键词: Binding; Biology; Cell Differentiation process; Cell Line; Cell Nucleus; Cell model; Cells; Chromatin; Chromosome Breakage; Clustered Regularly Interspaced Short Palindromic Repeats; Coupling; Cytoplasm; DNA; DNA Damage; DNA Double Strand Break; DNA Maintenance; DNA Repair; DNA Repair Gene; DNA Repair Pathway; DNA biosynthesis; Data; Defect; Disease; Down-Regulation; Enzymes; Etiology; Exhibits; FMR1; Fibroblasts; Fragile X Syndrome; Gene Expression; Genes; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Goals; Human; Hybrids; Impairment; In Vitro; Intellectual functioning disability; Knock-out; Light; Link; Maintenance; Messenger RNA; Modeling; Molecular; Mutate; Mutation; Neurodevelopmental Disorder; Neurons; Nuclear; Nuclear Protein; Pathway interactions; Patients; Phenotype; Play; Prevention; Protein Deficiency; Proteins; Proteome; RNA; RNA Helicase; Regulation; Reporting; Research; Research Project Grants; Research Proposals; Resolution; Resolvase; Role; Stress; Structure; Synapses; TP53 gene; Testing; Translational Regulation; Translations; autism spectrum disorder; cell type; disease phenotype; genome-wide; helicase; in vivo; induced pluripotent stem cell; loss of function; lymphoblast; mutant; nerve stem cell; neuron development; new therapeutic target; novel therapeutic intervention; preservation; prevent; recruit; replication stress; risk variant; therapeutic target; transcriptome; virtual

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.

项目摘要/摘要 拟议的研究项目的主要目标是了解FMRP的核功能 对脆弱X综合征（FXS）病因的影响。 FXS发生在FMR1中的突变时发生 基因导致FMRP功能的缺乏或丧失。 FMRP主要被视为翻译 细胞质中广泛的mRNA底物的阻遏物，但其核功能不是很好 理解。我们最近报道说，源自FXS患者的细胞患有全基因组DNA双链 在复制应力下时断裂（DSB）。此外，FXS细胞中的DNA DSB发生在附近 在基因转录过程中容易形成DNA的序列：称为R环的RNA杂种。这个发现 提出了FMRP在预防R-loop诱导的DSB中的新功能，从而 保持基因组稳定性。遵循此范式移动发现，我们发现FMRP直接结合R- 通过多价相互作用，循环和DHX9是R环分辨率。因此，我们的研究提供了 FMRP通过桥接R环和R环分辨率来协助R环分辨率的机制 染色质。此外，我们观察到在几乎所有DNA修复途径中的基因表达降低 FXS基因组，具有和没有复制应力，将此表型与受损的p53途径联系起来。 这项提出的研究我们将扩展分析，询问FMRP缺乏是否会导致全基因组DNA损伤 在人类神经元中。我们将系统地识别并比较神经元中的DNA DSB和R环 从FXS患者诱导多能细胞，通过这样做，我们将辨别出最多的“高风险”基因 在缺乏FMRP的细胞中容易受到DSB的影响和下调，从而提供了一个简短的潜力清单 FXS的治疗靶标。此外，我们已经观察到FMRP和DHX9之间的直接相互作用 RNA：DNA杂交解旋酶。我们将确定FMRP桥接R-loop和dhx9的机制 促进R环的分辨率。我们还将在人神经元中探测FMRP核蛋白质组，以鉴定 与染色质上与FMRP相互作用的其他因素。我们提议的项目将进一步 了解FMRP基因组维持功能，并有望将新的光浸入病因 FXS的基础。