Regulation of IL7R splicing by DDX39B and its role in Multiple Sclerosis

DDX39B 对 IL7R 剪接的调节及其在多发性硬化症中的作用

• 批准号: 9073091
• 负责人: Gaddiel Galarza Munoz
• 金额: $ 2.58万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-01 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9073091
• 关键词: Regulation; IL7R; splicing; DDX39B; its

Multiple Sclerosis (MS) is an autoimmune disorder of the central nervous system typified by axonal demyelination and neuronal death. To date, there is no effective treatment to cure the disease, and the available therapies do not alter the outcome of the disease. The development of better therapeutic options requires in-depth understanding of the molecular mechanisms leading to disease development and progression. Our group previously discovered a single nucleotide polymorphism (SNP, rs6897932) within exon 6 of the IL-7 receptor alpha chain (IL7R) that is strongly associated with the risk of developing MS. Our group further showed that the MS-associated allele of the SNP increases skipping of exon 6 both in vitro and in vivo, leading to increased production of a secreted receptor (sIL7R) that is unable to activate the IL-7 signaling pathway. Importantly, sIL7R has been linked to the disease in both human patients and animal models. These results directly implicate splicing of IL7R to the pathogenesis of MS, and posit the trans-acting factors controlling its splicing as candidate MS susceptibility genes. I have uncovered two of the trans-factors controlling IL7R exon 6 splicing: the RNA helicase DDX39B, which activates exon inclusion, and the polypyrimidine tract binding protein (PTBP1), which represses it. Furthermore, we uncovered several SNPs in the DDX39B gene region that are associated with MS, thereby establishing DDX39B is itself a risk factor for MS. None of the associated SNPs are located within the coding sequence of the gene, suggesting that one or more of these SNPs may contribute to the disease association by modifying DDX39B expression. Through bioinformatics and gene expression analyses, I have uncovered mRNA isoforms encoding either the full-length protein or a novel short protein, and several mRNA isoforms that are candidate targets for nonsense-mediated decay. Importantly, I have established two of the SNPs could alter expression levels of these isoforms. The goal of this proposal is to provide functional links connecting DDX39B to the pathogenesis of MS. Specifically, I aim to: 1) elucidate its role in the regulation of IL7R exon 6 splicing; 2) understand the functional roles of the different protein isoforms; and 3) uncover the SNPs responsible for its association with MS. I will combine biochemical and genetic approaches to elucidate the mechanism by which DDX39B activates exon 6 splicing, and to characterize the functional roles of the protein isoforms. I will test the impact of selected SNPs on DDX39B expression by combining in vivo gene expression analysis and functional studies using reporter minigenes. Successful completion of this research will advance our current understanding of the molecular underpinnings of MS, in particular by functionally linking DDX39B to the pathogenesis of MS, and providing a functional characterization of the DDX39B gene. Given that DDX39B has been associated with numerous autoimmune disorders, our results could be relevant to the mechanistic etiology of other autoimmune diseases.

多发性硬化症（MS）是一种中枢神经系统自身免疫性疾病，以轴突病变为代表 脱髓鞘和神经元死亡。迄今为止，尚无有效的治疗方法可以治愈该疾病，并且 现有的疗法不会改变疾病的结果。开发更好的治疗选择 需要深入了解导致疾病发生和发展的分子机制 进展。我们课题组之前在外显子内发现了一个单核苷酸多态性（SNP，rs6897932） IL-7 受体 α 链 (IL7R) 的 6 号与患 MS 的风险密切相关。我们组 进一步表明，SNP 的 MS 相关等位基因在体外和体内都会增加外显子 6 的跳跃， 导致无法激活 IL-7 信号传导的分泌受体 (sIL7R) 的产生增加 途径。重要的是，sIL7R 与人类患者和动物模型中的疾病有关。这些 结果直接表明 IL7R 剪接与 MS 的发病机制有关，并推测反式作用因子 控制其剪接作为候选 MS 易感基因。我发现了两个反式因子 控制 IL7R 外显子 6 剪接：RNA 解旋酶 DDX39B，可激活外显子包含，以及 多嘧啶束结合蛋白 (PTBP1) 会抑制它。此外，我们还发现了一些 SNP DDX39B 基因区域与 MS 相关，因此确定 DDX39B 本身就是 MS 的危险因素 多发性硬化症。相关的 SNP 均不位于该基因的编码序列内，这表明一个或多个 更多这些 SNP 可能通过修改 DDX39B 表达来促进疾病关联。通过 通过生物信息学和基因表达分析，我发现了编码全长的 mRNA 亚型 蛋白质或新型短蛋白质，以及几种作为无义介导候选靶标的 mRNA 亚型 衰变。重要的是，我已经确定其中两个 SNP 可以改变这些亚型的表达水平。 该提案的目标是提供 DDX39B 与 MS 发病机制之间的功能联系。 具体来说，我的目标是：1）阐明其在IL7R外显子6剪接调节中的作用； 2）了解函数式 不同蛋白质亚型的作用； 3) 揭示与 MS 相关的 SNP。我会 结合生化和遗传学方法阐明 DDX39B 激活外显子 6 的机制 剪接，并表征蛋白质亚型的功能作用。我将测试所选 SNP 的影响 通过结合体内基因表达分析和使用报告基因的功能研究来研究 DDX39B 表达 小基因。这项研究的成功完成将增进我们目前对分子生物学的理解。 MS 的基础，特别是通过在功能上将 DDX39B 与 MS 的发病机制联系起来，并提供 DDX39B 基因的功能表征。鉴于 DDX39B 已与众多 自身免疫性疾病，我们的结果可能与其他自身免疫性疾病的机制病因学相关。