Regulation of Rhythmic m6A RNA Modification by ER‐associated Degradation

内质网（ER）相关降解（ERAD）是一个主要的ER质量控制程序， 监测和易位未折叠或错误折叠的蛋白质底物从ER到胞质溶胶的多聚泛素化 和蛋白酶体降解。N6-甲基腺苷（m6 A）甲基化，最普遍的内部修饰 已知哺乳动物mRNAs的基因调控几乎每一个主要类别的基因的稳定性、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 m6 A甲基化过程。然而，信号传导途径 潜在的RNA m6 A修饰的调控仍然难以捉摸。在此，我们积累了强大的初步 一个前所未有的昼夜节律调节ERAD途径控制mRNA m6 A修饰的证据， 随后的脂质稳态，我们称之为“昼夜ERAD-m6 A”。我们的主要初步研究结果包括： (i)ER-驻留的E3泛素连接酶HRD 1及其辅因子SEL 1 L，ERAD机制的主要组分， 由肝脏中的生物钟调节;（ii）HRD 1与多聚泛素化相互作用并介导多聚泛素化， 特异性m6 A写入器L14和读取器YTHDF 3的降解;（iii）HRD 1肝脏特异性KO（LKO） 小鼠在肝m6 A mRNA中显示与胃L14-LKO或YTHDF 3敲低小鼠相反的方式 甲基化水平、脂质代谢调节因子的表达以及与肝硬化相关的代谢表型。 脂肪变性和高脂血症;和（iv）与经典ERAD不同，新鉴定的ERAD-m6 A调节轴 其在肝脏脂质代谢中的作用受昼夜节律的控制。这些观察结果导致 我们的中心假设是肝脏HRD 1-ERAD程序，它在生物钟下振荡， 通过控制特异性m6 A写入器L14的节律性降解来调节肝m6 A RNA修饰 阅读器YTHDF 3这种前所未有的昼夜ERAD-m6 A RNA修饰调控网络， 可能是由昼夜节律干扰线索失调，代表了一个主要的途径，控制代谢 与肝脂肪变性和高脂血症相关的体内平衡。 在这项应用中，我们将利用分子和细胞的方法，基因工程动物模型， 和m6 A RNA修饰的高通量分析，以关键性地解决功能和机制， 昼夜ERAD调节肝脏m6 A RNA修饰和脂质代谢。在两个目标中，我们将：1）定义一个 新的昼夜ERAD途径，通过降解特异性的 m6 A写入器和读取器;以及2）确定昼夜ERAD-m6 A RNA修饰的功能意义 维持脂质稳态的途径。在这个项目完成后，我们将揭示的功能， 一种新的昼夜ERAD-m6 A RNA修饰途径调节脂质稳态的机制 与代谢紊乱有关这一发现将为生理学的研究开辟新的范式 ERAD和m6 A RNA修饰，为开发代谢性疾病的治疗方法提供了新的思路。

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.