Regulation of Rhythmic m6A RNA Modification by ER‐associated Degradation

内质网(ER)相关降解(ERAD)是一项主要的ER质量控制计划， 监测并将未折叠或错误折叠的蛋白质底物从内质网转移到胞浆中进行多泛素化 和蛋白酶体的降解。N6-甲基腺苷(M6A)甲基化，最普遍的内部修饰 在哺乳动物的mRNAs中，已知调节几乎所有主要类别的mRNA的稳定性、翻译和功能。 人类的RNA。三个主要的蛋白质家族，包括编写器、阅读器和擦除器，是已知的 负责可逆的RNA m6A甲基化过程。然而，信号转导途径 在RNA M6A的调控下，修饰仍然难以捉摸。在此，我们积累了强大的初步经验 一种史无前例的昼夜节律调节的ERAD途径控制mRNAm6A修饰和 随后的脂平衡，我们称之为“昼夜ERAD-M6A”。我们的主要初步发现包括： (I)ERAD机制的主要组成部分--内质网驻留的E3泛素连接酶Hrd1及其辅助因子SEL1L， 由肝脏的生物钟调节；(Ii)Hrd1与多泛素相互作用并介导多泛素化和 特异性m6A写入物METTL14和读取物YTHDF3的降解； METTL14-LKO或YTHDF3基因敲除小鼠肝脏M6A基因表达出现逆转 与肝脏相关的甲基化水平、脂代谢调节剂的表达和代谢表型 与经典的ERAD不同，新发现的ERAD-m6A调节轴 其在肝脂代谢中的作用受昼夜节律的控制。这些观察结果导致了 我们的中心假设是肝脏Hrd1-ERAD程序在昼夜节律下振荡， 通过控制特定m6A编写器METTL14的节律性降解来调节肝脏m6A RNA的修饰 和阅读器YTHDF3。这个史无前例的昼夜节律ERAD-M6A RNA修饰调控网络， 可能受到扰乱昼夜节律的信号的失调，代表着控制新陈代谢的主要途径 与肝脏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方法，遗传工程动物模型， 以及高通量分析m6A RNA修饰，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。在两个目标中，我们将：1)定义 新的昼夜节律ERAD途径通过降解特定的M6A RNA来调节节律性的M6A RNA修饰 M6A写入者和读取者；以及2)确定昼夜ERAD-m6A RNA修饰的功能意义 维持脂类平衡的途径。在这个项目完成后，我们将揭示其功能和 一种新的昼夜节律ERAD-M6A RNA修饰途径调节脂质稳态的机制 与代谢紊乱有关。这一发现将为生理学研究开辟新的范式。 Erad和m6A的RNA修饰，为开发代谢性疾病的治疗方法提供了新的线索。

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.