Interrogating the role of m6A mRNA methylation in the aging of the β-cell and diabetes

项目总结 虽然1型糖尿病(T1D)和2型糖尿病(T2D)的病因不同，但减少了 功能性β细胞团是这两种疾病的中心特征。协调一致的研究努力旨在开发 改善糖尿病患者β细胞存活率和恢复β细胞功能的策略。细胞衰老是 这是衰老的一个主要标志。最近的研究表明，β细胞衰老是导致 1型糖尿病(T1D)和2型糖尿病(T2D)。RNA修饰正在成为重要的调节因子 衰老的许多特征。我们已经发现m6amRNA甲基化对β细胞的功能和 生存，并最近表征了人类T1D和T2D胰岛的M6A景观。我们的初步数据 显示m6A擦除器ALKBH5在衰老的人β细胞中表达上调，并且m6A差异甲基化 与对照组相比，T1D和T2D中的基因对DNA损伤反应和衰老途径具有丰富的作用。 本申请的目的是使用最先进的方法来表征老化的β的m6A甲基组- 并确定糖尿病中β细胞加速衰老的基础上的m6A调节通路。我要测试一下 主要假设衰老的β细胞表现出转录特异性的m6A低甲基化和随之而来的 参与推动细胞衰老的mRNAs上调，因此m6A是一种有价值的治疗方法 糖尿病的治疗目标。在特定的目标1中，我将描述老化的β细胞的时间m6A景观。 目标1将在K99阶段完成，这将为我提供老化方面的培训 独立完成m6A数据的生物学和生物信息学分析的进一步经验 R00阶段和未来的R01提交。在跨越K99和R00阶段的具体目标2中，我将 确定ALKBH5在人β细胞向衰老转变过程中的分子靶点。完成分项工作- 在K99阶段提出的目标2的目标将为我提供光活化核糖核苷的培训- 增强的交联和免疫沉淀(PAR-CLIP)测序和数据分析 补充目标1，并在R00阶段独立完成目标2。对于具体的目标3，我将瞄准 体内M6A水平可减少β细胞衰老，改善糖尿病。完成这一目标将使我能够 验证在AIM的1和2中确定的候选基因，并在体内测试Alkbh5在驱动加速的 糖尿病患者β细胞出现衰老。这些实验是新颖的，因为它们结合了基于组学的方法， 小鼠遗传模型、生理学和分子生物学，以检验m6A的机制作用，以及 特别是ALKBH5，在糖尿病的β-细胞衰老转变中。

PROJECT SUMMARY Although the etiopathogenesis of Type 1 (T1D) and Type 2 diabetes (T2D) is different, decreased functional β-cell mass is a central feature in both diseases. Concerted research efforts aim at developing strategies to improve β-cell survival and restore β-cell function in patients with diabetes. Cellular senescence is a major hallmark of aging. Recent work has demonstrated that β-cell senescence is a common contributor to Type 1 diabetes (T1D) and Type 2 diabetes (T2D). RNA modifications are emerging as important modulators of many hallmarks of aging. We have discovered that m6A mRNA methylation is essential for β-cell function and survival, and recently characterized the m6A landscape of human T1D and T2D islets. Our preliminary data shows that the m6A eraser ALKBH5 is upregulated in aged human β-cells and differentially m6A methylated genes in T1D and T2D are enriched for DNA damage response and senescence pathways compared to controls. The goal of this application is to use state-of-the art methods to characterize the m6A methylome of the aging β- cell and identify m6A-regulated pathways that underlie the accelerated β-cell senescence in diabetes. I will test the overarching hypothesis that aging β-cells exhibit transcript-specific m6A hypomethylation and consequent upregulation of mRNAs involved in driving cell senescence, and that m6A is therefore a valuable therapeutic target for diabetes. In Specific Aim 1, I will characterize the temporal m6A landscape of the aging β-cell. Completion of Aim 1, which will take place during K99 phase, will provide me with training in aspects of aging biology and further experience with bioinformatics analysis of m6A data necessary to independently complete the R00 phase and future R01 submissions. In Specific Aim 2, which will span the K99 and R00 phases, I will identify the molecular targets of ALKBH5 in the human β-cell transition to senescence. Completion of the sub- aim of Aim 2 proposed during the K99 phase will provide me with the training in photoactivatable ribonucleoside- enhanced crosslinking and immunoprecipitation (PAR-CLIP) sequencing and data analysis necessary to complement Aim 1, and independently complete Aim 2 during the R00 phase. For Specific Aim 3, I will target m6A levels to reduce β-cell senescence and improve diabetes in vivo. Completion of this aim will allow me to validate candidate genes identified in Aim’s 1 and 2 and to test in vivo the role of Alkbh5 in driving the accelerated senescence seen in β-cells in diabetes. These experiments are novel as they combine omics-based approaches, mouse genetic models, physiology, and molecular biology to examine the mechanistic role of m6A, and particularly ALKBH5, in β-cell senescence transition in diabetes.