Alternative polyadenylation as a novel mechanism for diabetes

项目摘要 肥胖和相关的胰岛素抵抗是2型糖尿病（T2 D）的既定危险因素。虽然 超过90%的T2 D患者超重或肥胖，仅约30%的肥胖者发展为T2 D。一 肥胖患者发生T2 D的主要决定因素是胰岛β细胞分解或衰竭， 导致胰岛素相对缺乏。据文献记载，胰岛功能衰竭有很强的遗传倾向。 全基因组关联研究（GWAS）为关联遗传变异（SNP）与T2 D提供了有力的工具。 然而，绝大多数来自GWAS的糖尿病风险SNPs发现于非编码区， 识别T2 D SNP相关基因的重大挑战。3 '非翻译区（3' UTR）是非翻译区。 含有顺式调节元件（CRE）的编码序列，例如miRNA和RNA结合位点 蛋白质（RBP）调节mRNA命运和蛋白质表达。3 'UTR处的交替多聚腺苷酸化（阿帕） 是一种RNA加工机制，其产生具有显著不同的3 'UTR长度的mRNA同种型， 不同的克雷斯。调节阿帕的SNPs可以延长或缩短3 'UTR，从而改变基因表达， 功能我们最近开发了一种新的3 'UTR阿帕数量性状基因座（3' aQTLs）分析工具， 与阿帕疾病相关的SNP。利用3 'aQTL工具，我们发现锌的3' UTR的延长 胰腺中的指状CCCH结构域蛋白13（ZC 3 H13）与T2 D高度相关。ZC3H13 是一种关键的表位转录组因子，形成N6-甲基腺苷（m6 A）RNA修饰写入器复合物， 甲基转移酶样3（methyltransferase-like 3，简称为L3）和甲基转移酶样14（methyltransferase-like 14，简称为L14）。胃L3和胃L14- 已经显示，介导的m6 A对于β细胞功能是必需的。我们的初步数据显示， ZC 3 H13 3 'UTR降低ZC 3 H13蛋白表达而不改变mRNA水平。打倒ZC 3 H13 抑制培养的β细胞中的胰岛素产生。此外，ZC 3 H13杂合敲除小鼠 当用高脂饮食挑战时，表现出葡萄糖耐量受损和胰岛素水平降低。我们， 因此，假设来自3 'aQTL相关的3' UTR的ZC 3 H13蛋白表达减少， 延长有助于T2 D中胰岛衰竭的遗传倾向。我们提出三个目标，研究什么 SNP导致ZC 3 H13 3 'UTR延长，为什么ZC 3 H13 3' UTR延长降低蛋白质表达， 减少的ZC 3 H13蛋白如何损害胰岛功能。目的1：确定影响阿帕的因果SNP， ZC 3 H13中使用3 'aQTL的功能信息精细定位。目标2：确定 3 'UTR延长降低ZC 3 H13蛋白表达。目的3：研究 降低的ZC 3 H13表达损害胰岛素产生和胰岛功能。我们的研究将推动该领域的发展， 揭示ZC 3 H13阿帕作为一种新的遗传风险因子，并阐明ZC 3 H13介导的表观转录组学 调节作为T2 D中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.