Regulation of Rhytmic m6A RNA Modification by ER associated Degradation

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, writers, readers, and erasers, are 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, the major component of ERAD machinery, is 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; and (iii) HRD1 liver-specific KO (LKO) mice are opposite to METTL14-LKO or YTHDF3-knockdown (KD) mice in hepatic m6A mRNA modification levels, expression of lipid metabolic regulators, and metabolic phenotypes associated with hepatic steatosis and hyperlipidemia. 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, as well as high throughput profiling of m6A RNA modification to critically address the function and mechanism by which circadian ERAD regulates 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 an unprecedented circadian ERAD-m6A RNA modification pathway regulates lipid homeostasis associated with metabolic disorders. The findings will open new paradigms for the studies on the physiological ERAD and m6A RNA modification, and shed new light on developing therapeutics for lipid-associated metabolic disease.