Regulation of Rhythmic m6A RNA Modification by ER‐associated Degradation

ERα相关降解对节律性 m6A RNA 修饰的调节

• 批准号: 10454294
• 负责人: Deyu Fang
• 金额: $ 55.86万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-16 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10454294
• 关键词: Address; Animal Model; Apoptosis; Binding; Cellular Metabolic Process; Circadian Rhythms; Cues; Cytosol; Endoplasmic Reticulum; Endoplasmic Reticulum Degradation Pathway; Enzymes; Fatty Liver; Genetic Engineering; Genetic Translation; Goals; Hepatic; High Fat Diet; Homeostasis; Human; Hyperlipidemia; Knockout Mice; Light; Lipids; Liver; Mediating; Messenger RNA; Metabolic; Metabolic Control; Metabolic Diseases; Metabolism; Methylation; Modification; Molecular; Monitor; Mus; Pathway interactions; Periodicity; Physiological; Physiological Processes; Play; Polyubiquitination; Process; Protein Family; Proteins; Quality Control; RNA; Reader; Regulation; Risk; Role; Signal Transduction Pathway; Testing; Therapeutic; Time-restricted feeding; Translations; Ubiquitination; circadian; circadian pacemaker; knock-down; lipid metabolism; mRNA Stability; metabolic phenotype; misfolded protein; non-alcoholic fatty liver disease; novel; preservation; programs; ubiquitin-protein ligase

Endoplasmic Reticulum (ER)-Associated Degradation (ERAD) is a major ER quality-control program that monitors and translocates unfolded or misfolded protein substrates from the ER to cytosol for polyubiquitination and proteasomal degradation. N6-methyladenosine (m6A) methylation, the most prevalent internal modification of mammalian mRNAs, is known to regulate the stability, translation, and function of almost every major class of human RNAs. Three major families of proteins, including writers, readers, and erasers, are known to be responsible for the reversible RNA m6A methylation process. However, the signal transduction pathway underlying the regulation of RNA m6A modification remain elusive. Herein, we accumulated strong preliminary evidence for an unprecedented circadian-regulated ERAD pathway that controls mRNA m6A modification and subsequent lipid homeostasis, which we called “circadian ERAD-m6A”. Our major preliminary findings include: (i) the ER-resident E3 ubiquitin ligase HRD1 and its co-factor SEL1L, the major components of ERAD machinery, are regulated by the circadian clock in the liver; (ii) HRD1 interacts with and mediates polyubiquitination and degradation of the specific m6A writer METTL14 and the reader YTHDF3; (iii) HRD1 liver-specific KO (LKO) mice display reversed fashions with METTL14-LKO or YTHDF3-knockdown mice in hepatic m6A mRNA methylation levels, expression of lipid metabolic regulators, and metabolic phenotypes associated with hepatic steatosis and hyperlipidemia; and (iv) unlike the classic ERAD, the newly-identified ERAD-m6A regulatory axis and its function in hepatic lipid metabolism are under the control of circadian rhythm. These observations led to our central hypothesis that the liver HRD1-ERAD program, which is oscillated under the circadian clock, regulates hepatic m6A RNA modification by controlling rhythmic degradation of the specific m6A writer METTL14 and the reader YTHDF3. This unprecedented circadian ERAD-m6A RNA modification regulatory network, which may be dysregulated by circadian-disrupting cues, represents a major pathway that controls metabolic homeostasis associated with hepatic steatosis and hyperlipidemia. In this application, we will utilize molecular and cellular approaches, genetically engineered animal models, and high-throughput profiling of m6A RNA modification to critically address the function and mechanism by which circadian ERAD regulates hepatic m6A RNA modification and lipid metabolism. In two aims, we will: 1) define a novel circadian ERAD pathway that modulates rhythmic m6A RNA modification through degrading the specific m6A writer and reader; and 2) determine the functional significance of circadian ERAD-m6A RNA modification pathway in maintaining lipid homeostasis. Upon completion of this project, we will reveal the function and mechanism by which a novel circadian ERAD-m6A RNA modification pathway regulates lipid homeostasis associated with metabolic disorders. The findings will open up new paradigms for the studies on the physiological ERAD and m6A RNA modification and shed new light on developing therapeutics for metabolic disease.

内质网 (ER) 相关降解 (ERAD) 是一项主要的 ER 质量控制程序， 监测未折叠或错误折叠的蛋白质底物并将其从内质网转移到胞质溶胶以进行多泛素化 和蛋白酶体降解。 N6-甲基腺苷 (m6A) 甲基化，最常见的内部修饰 众所周知，哺乳动物 mRNA 可以调节几乎所有主要类别的稳定性、翻译和功能。 人类RNA。已知蛋白质的三个主要家族，包括写入器、读取器和擦除器 负责可逆的 RNA m6A 甲基化过程。然而，信号转导途径 RNA m6A 修饰的调控机制仍然难以捉摸。在此，我们积累了雄厚的前期基础 史无前例的昼夜节律调节 ERAD 通路控制 mRNA m6A 修饰的证据 随后的脂质稳态，我们称之为“昼夜节律ERAD-m6A”。我们的主要初步发现包括： (i) ER驻留E3泛素连接酶HRD1及其辅因子SEL1L，ERAD机器的主要组成部分， 受肝脏生物钟调节； (ii) HRD1 与多泛素化相互作用并介导 特定 m6A 写入器 METTL14 和读取器 YTHDF3 退化； (iii) HRD1 肝脏特异性 KO (LKO) METTL14-LKO 或 YTHDF3 敲低小鼠的肝脏 m6A mRNA 表现出相反的模式 甲基化水平、脂质代谢调节因子的表达以及与肝相关的代谢表型 脂肪变性和高脂血症； (iv) 与经典的 ERAD 不同，新确定的 ERAD-m6A 调控轴 其在肝脏脂质代谢中的功能受昼夜节律的控制。这些观察导致 我们的中心假设是，肝脏 HRD1-ERAD 程序在生物钟下振荡， 通过控制特定 m6A writer METTL14 的节律性降解来调节肝脏 m6A RNA 修饰 和读者YTHDF3。这种前所未有的昼夜节律 ERAD-m6A RNA 修饰调控网络， 可能会因昼夜节律干扰信号而失调，代表控制代谢的主要途径 与肝脂肪变性和高脂血症相关的稳态。 在此应用中，我们将利用分子和细胞方法、基因工程动物模型、 以及 m6A RNA 修饰的高通量分析，以关键地解决其功能和机制 昼夜节律 ERAD 调节肝脏 m6A RNA 修饰和脂质代谢。为了实现两个目标，我们将：1）定义 新型昼夜节律 ERAD 通路通过降解特定的 m6A RNA 修饰来调节节律性 m6A RNA 修饰 m6A 作家和读者； 2) 确定昼夜节律 ERAD-m6A RNA 修饰的功能意义 维持脂质稳态的途径。当这个项目完成后，我们将揭示其功能和 新型昼夜节律 ERAD-m6A RNA 修饰途径调节脂质稳态的机制 与代谢紊乱有关。该研究结果将为生理学研究开辟新范式 ERAD 和 m6A RNA 修饰为开发代谢疾病疗法提供了新的思路。