RNA在FUS介导的神经细胞DNA损伤修复中的作用

FUS is a DNA/RNA binding protein that was recently linked to fALS and FTD. Our previous studies found that FUS is important for DNA damage response (DDR), and implementation of this function involves a direct interaction with histone deacetylase 1 (HDAC1). Remarkably, FUS carrying familial ALS (fALS) mutations are defective in DNA repair, and transgenic mice expressing fALS FUS-R521C mutant proteins exhibit increased DNA damage in cortical and spinal motor neurons. The similar results were also recapitulated in brain sections from familiar ALS patients harboring FUS mutations. Moreover, recent studies have revealed the involvement of the RNA processing/binding proteins and regulatory non-coding RNA (ncRNAs) in the DDR. Based on these previous observations, we plan to investigate whether the RNAs that bind to FUS are involved in (DDR). My goals for this project are to test the hypothesis that ncRNAs may function as guide molecules directing the recruitment of FUS to DSB sites to facilitate repair, and to determine the impact of these RNAs to the onset and development of ALS and FTLD. This project involves interdisciplinary approaches with collaborations between computational biologists, molecular biologists, and neurobiologists, both within the lab and with other labs. We will use RNA-seq to determine the regulatory RNAs that bind to FUS in response to DNA damage. Top candidates obtained from RNA-seq will be further evaluated using a cell-based GFP reporter assay to determine its impact on DDR and neuronal genome stability. We will investigate how the interaction of FUS/RNA are regulated in response to DNA damage using chromatin immunoprecipitation (ChIP), in vitro gel mobility shift assay, cell based reporter assay, and laser micro-irradiation assay. We will investigate whether mutations of FUS exhibit deficits in its association with RNAs in response to DNA damage, and how this contributes to the pathogenesis of ALS/FTLD. We believe that successful finish of this project will provide us a better understanding of the cellular and molecular mechanisms of diseases with the potential to screen and identify novel therapeutic targets.

FUS是一个DNA/RNA结合蛋白，是脊髓侧索硬化证(ALS)的致病基因之一。我们最新的研究发现, FUS在神经细胞的DNA损伤修复中起着非常重要的作用,更重要的是FUS的ALS致病突变体的DNA损伤修复功能缺失，提示DNA损伤应答和修复很有可能是含有FUS突变的fALS的发病机理。 然而，与FUS相结合的RNA是否参与其在DNA损伤修复中的作用和FUS/RNA相互作用在ALS发病中的作用未知。因此，本研究课题将着眼于研究与FUS结合的RNAs在DNA损伤损伤中的作用及其机制。通过这项研究，我们希望找到可以将DNA损伤修复蛋白(例如FUS)招募至DNA损伤部位的非编码RNA，并且阐述其在DNA损伤应答反应和FUS突变引起的ALS中的作用机制。这一研究项目的完成将会促进我们对于FUS功能异常所造成的神经退行性疾病的发病机理的理解，同时也为RNA在DNA损伤应答机制中的作用提供新的思路。

脊髓侧索硬化（Amyotrophic Lateral Sclerosis ,ALS）是一种以运动皮层，脑干和脊髓运动神经元渐进死亡为主要特征的神经退行性疾病，目前为止，其发病机制还不是很清楚。DNA/RNA结合蛋白FUS是近年新发现的ALS的致病基因之一，我们前期的研究表明FUS通过和HDAC1相互作用调控神经系统基因组DNA的损伤修复，ALS致病性FUS突变体可以正常的感应对基因组DNA的损伤，但修复蛋白复合体不能完整的组装。然而FUS所结合的RNA是否参与到其所介导的DNA损伤反应中未知。在本课题中，我们首先利用HITS-CLIP高通量测序的方法发现FUS可以结合脑特异性高表达的非编码RNA Mir9-3hg，我们还鉴定到了三个Mir-3hg在脑中高表达的新的转录本。然后我们用一系列的生化和细胞实验证明Mir-3hg在DNA的损伤修复中起着非常重要的作用。我们接着用CHIRP的方法鉴定到miR9-3hg和HMGB1具有相互作用。miR9-3g 的基因缺失小鼠HMGB的表达显著降低，HMBG1基因敲底的神经细胞其DNA损伤修复的能力显著降低。目前我们的结果表明HMBG1，FUS和miR9-3hg组成一个复合物在DNA损伤修复中协同作用发挥调节功能。这项研究课题的完成将有助于我们深入了解lncRNA在神经系统基因组DNA损伤修复中及神经退化性疾病中的作用。