Alternative polyadenylation as a novel mechanism for diabetes

项目总结 肥胖和与之相关的胰岛素抵抗是2型糖尿病(T2D)的既定危险因素。虽然 超过90%的T2D患者超重或肥胖，只有大约30%的肥胖者发展为T2D。一 肥胖患者发生T2D的主要决定因素是胰岛β−细胞分解或衰竭， 导致相对的胰岛素缺乏。已有文献证明胰岛功能衰竭具有很强的遗传易感性。 全基因组关联研究为遗传变异(SNPs)与T2D的关联提供了强有力的工具。 然而，来自GWAS的绝大多数糖尿病风险SNPs都是在非编码区发现的， 识别T2D的SNP相关基因面临重大挑战。3‘非翻译区域(3’非翻译区域)是非 编码含有顺式调节元件(Cre)的序列，如miRNAs结合位点和RNA结合 调节mRNA命运和蛋白表达的蛋白质(RBPs)。3‘非编码区的交替多聚腺苷(APA) 是一种RNA加工机制，它产生具有显著不同的3‘非编码区长度的信使核糖核酸亚型 截然不同的Cres。调节APA的SNPs可能延长或缩短3‘非编码区，从而改变基因表达和 功能。我们最近开发了一种新的3‘UTRAPA数量性状基因座(3’aQTL)分析工具，用于协同定位 与APA相关的疾病相关SNPs。利用3‘aQTL工具，我们发现锌的3’非编码区的延长 胰腺中的Finger CCCH结构域蛋白13(ZC3H13)与T2D密切相关。ZC3H13 是一个关键的表位转录因子，它与N6-甲基腺苷(M6A)RNA修饰写入物形成复合体 甲基转移酶样3(METTL3)和甲基转移酶样14(METTL14)。METTL3和METTL14- 已有研究表明，介导的m6A对β细胞的功能是必不可少的。我们的初步数据显示，较长时间 ZC3H13 3‘非编码区减少ZC3H13蛋白表达，但不影响其基因表达水平。击倒ZC3H13 抑制培养的β-细胞产生胰岛素。此外，ZC3H13杂合基因敲除小鼠 当受到高脂肪饮食的挑战时，表现出葡萄糖耐量受损，胰岛素水平降低。我们, 因此，假设3‘aQTL相关的3’非编码区ZC3H13蛋白表达降低 在T2D中，延长与胰岛衰竭的遗传易感性有关。我们提出三个目标来研究 SNP导致ZC3H13 3‘非编码区延长，为什么ZC3H13 3’非编码区延长会降低蛋白质表达，以及 降低的ZC3H13蛋白如何损害胰岛功能。目的1：确定影响APA的SNPs ZC3H13利用3‘aQTL的功能信息精细定位。目标2：确定通过哪些机制 3‘端非编码区延长可降低ZC3H13蛋白表达。目的3：探讨其作用机制 ZC3H13表达减少会损害胰岛素的产生和胰岛功能。我们的研究将推动这一领域的发展 ZC3H13 APA作为新的遗传危险因子的发现及ZC3H13介导的表位转录 调节是T2D患者β细胞功能受损的新机制。

PROJECT SUMMARY Obesity and the associated insulin resistance are the established risk factors for type 2 diabetes (T2D). Although more than 90% of T2D patients are overweight or obese, only about 30% of obese people develop T2D. One major determining factor for the development of T2D in obese patients is islet β−cell decomposition or failure, resulting in relative insulin deficiency. It is well documented that islet failure has a strong genetic predisposition. Genome-wide association study (GWAS) provides a powerful tool to associate genetic variants (SNPs) with T2D. However, the vast majority of the diabetes risk SNPs from GWAS are found in the non-coding regions, posing significant challenges to identifying the SNP-associated genes for T2D. 3’untranslated regions (3’UTR) are non- coding sequences containing cis-regulatory elements (CRE), such as binding sites for miRNAs and RNA-binding proteins (RBPs) that regulate mRNA fate and protein expression. Alternative polyadenylation (APA) at the 3’UTR is an RNA-processing mechanism that generates mRNA isoforms with significantly different 3’UTR lengths with distinct CREs. SNPs that regulate APA may lengthen or shorten 3’UTR, thereby altering gene expression and function. We recently developed a novel 3’UTR APA quantitative trait loci (3’aQTLs) analysis tool to colocalize disease-associated SNPs with APA. Using the 3’aQTLs tool, we found that the lengthening of 3’UTR of zinc finger CCCH domain-containing protein 13 (ZC3H13) in the pancreas was highly associated with T2D. ZC3H13 is a key epitranscriptomic factor that forms an N6-methyladenosine (m6A) RNA modification writer complex with methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14). Both METTL3 and METTL14- mediated m6A have been shown to be essential for β-cell function. Our preliminary data show that longer ZC3H13 3’UTR reduced ZC3H13 protein expression without changes to mRNA levels. Knocking down ZC3H13 suppressed insulin production in the cultured β-cells. Furthermore, ZC3H13 heterozygous knockout mice exhibited impaired glucose tolerance with reduced insulin levels when challenged with a high-fat diet. We, therefore, hypothesize that the reduced ZC3H13 protein expression from 3’aQTLs-associated 3’UTR lengthening contributes to the genetic predisposition of islet failure in T2D. We propose three aims to study what SNPs cause ZC3H13 3’UTR lengthening, why ZC3H13 3’UTR lengthening reduces protein expression, and how the reduced ZC3H13 protein impairs islet function. Aim 1: To identify the causal SNPs impacting APA of ZC3H13 using functionally informed fine-mapping of 3'aQTLs. Aim 2: To determine the mechanisms by which 3’UTR lengthening reduces ZC3H13 protein expression. Aim 3: To investigate the mechanisms by which reduced ZC3H13 expression impairs insulin production and islet function. Our studies will advance the field by uncovering ZC3H13 APA as a novel genetic risk factor and elucidating ZC3H13-mediated epitranscriptomic regulation as a novel mechanism for the impaired β-cell function in T2D.