Investigating the role of alternative splicing in the islets of Langerhans in developing diabetes.

来自小鼠的胰岛的转录组分析显示，这两种小鼠对糖尿病的易感性不同，如糖尿病易感的新西兰肥胖小鼠（NZO）和糖尿病抗性小鼠B6-ob/ob，这两种小鼠品系之间存在显著的表达差异，并发现了糖尿病基因。我们认为，差异基因表达不是糖尿病抵抗和易感性的唯一贡献者，剪接产生的不同蛋白质异构体可以影响胰岛的功能。这项提案的目的是研究和了解选择性剪接在糖尿病发病中的作用。因此，分析了来自雄性NZO和B6-ob/ob小鼠的分离胰岛的RNA-seq数据，并鉴定了730个具有可变剪接事件的基因。这一初步分析的结果将在三个主要工作计划中进一步研究：1.应鉴定并表征可能导致胰岛功能障碍的选择性剪接亚型。它将分析替代亚型如何不同于原始亚型，如果功能结构域或蛋白质的细胞内定位被改变，以及这如何影响胰岛素分泌等基本过程。总的来说，NZO外显子表现出更大的跳跃率，导致40%的移码和55%的框架保留变体，代表潜在改变的蛋白质。2.应确定可能影响选择性剪接的遗传变异。来自两种不同小鼠品系的RNAseq数据的分析揭示了大量的单核苷酸多态性（SNP），其可能有助于观察到的剪接模式。因此，这些SNPs的定位以及对调控元件的潜在影响有待进一步分析。将从GWAS数据中识别出的对选择性剪接有影响的SNP与人类糖尿病相关SNP进行比较，以便将我们的结果转化为人类数据。3.应分析胰岛中神经元特征的丧失如何与糖尿病的发作相关。来自胰岛的细胞显示出与神经元细胞相似的表达模式，并表达神经元富集的剪接因子如Srrm 4（丝氨酸/精氨酸重复基质4）。Srrm 4在来自NZO小鼠的胰岛中表达较低，并且已知的SRRM 4调节的外显子显示较高的跳跃。因此，将分析这些外显子如何改变蛋白质，从而改变β细胞在增殖、凋亡和胰岛素分泌等过程中的功能。

Transcriptome analysis of Islets of Langerhans derived from mice that differ in their susceptibility to develop diabetes, like the diabetes susceptible new zealand obese mouse (NZO) and the diabetes resistant mouse B6-ob/ob, revealed significant expression differences between the two mouse strains and the discovery of diabetes genes. We propose that differential gene expression is not the only contributor to diabetes resistance and susceptibility and that diverse protein isoforms resulting from splicing can influence the functionality of islets. The goal of this proposal is to study and understand the role of alternative splicing in the onset of diabetes. Therefore, RNA-seq data from isolated islets of male NZO and B6-ob/ob mice were analyzed and 730 genes with alternative splicing events identified. The results from this preliminary analysis shall be further studied in three major working plans: 1. Alternative splicing isoforms that might contribute to a dysfunction of the islets of Langerhans shall be identified and characterized. It will be analyzed how alternative isoforms differ from the original isoform, if functional domains or intracellular localization of the proteins are altered and how this can affect fundamental processes like insulin secretion. Overall NZO exons exhibit a larger skipping rate resulting in 40% frameshift and 55% frame preserving variants representing potentially altered proteins. 2. Genetic variants that might affect alternative splicing shall be identified. The analysis of RNAseq data from the two different mouse strains revealed a high number of single nucleotide polymorphisms (SNPs) that potentially contribute to the observed splicing pattern. Therefore, the localization of these SNPs and a potential impact on regulatory elements shall be further analyzed. Identified SNPs with impact on alternative splicing will be compared with human diabetes-associated SNPs from GWAS data in order to translate our results to human data. 3. It shall be analyzed how the loss of a neuronal signature in the islets of Langerhans can be associated with the onset of diabetes. Cells from islets show a similar expression pattern like neuronal cells and express neuronal enriched splicing factors like Srrm4 (serine/arginine repetitive matrix 4). Srrm4 is lower expressed in islets from NZO mice and known SRRM4 regulated exons show higher skipping. Therefore, it will be analyzed how these exons can alter proteins and thereby β-cell functionality with regard on processes like proliferation, apoptosis and insulin secretion.