Ubiquitin Ligase UBR5 Regulates Circadian Rhythm By Promoting The Ubiquitination And Degradation Of The Transcription Factor BMAL1 And Its Transcriptional Activity

Background and Aims:Circadian clock is the inner rhythm of life activities and controlled by a self-sustained and endogenous clock,which maintains the internal cycle of about 24 h.It is phylogenetically ubiquitous and exhibits at molecular,cellular,physiological,and behavioral levels.Circadian clock makes the life activities and behaviors of the organisms have obvious cyclical changes in the day and night.Several studies have found the importance of ubiquitin-proteasome system(UPS)in the regulation of circadian system.The core clock protein,aryl hydrocarbon receptor nuclear translocator-like protein 1(ARNTL or BMAL1),is the master regulator of the circadian clock and plays important roles in the regulation of many biological processes.BMAL1 is regulated by ubiquitin-proteasome system(UPS),but only few enzymes in the UPS were reported to regulate BMAL1 stability and affect circadian rhythm.Here,we screened the BMAL1 interacting partners through quantitative proteomics and discovered an E3 ubiquitin ligase which regulates the stability and biological function of BMAL1.Methods:The BMAL1 interacting partners were first discovered by mass spectrometric analysis.Immunoprecipitation,immunoblotting,and immunofluorescence experiments were used to validate the interaction.Second,we transfected UBR5 plasmid or siUBR5 into HEK293T cells or knocked out UBR5 in U20S cells to explore the effect of UBR5 on BMAL1 protein level.Third,we used proteasome inhibitor MG132 and cycloheximide(CHX)to explore the regulation of UBR5 on the stability and ubiquitination of BMAL1.Next,we used dual-luciferase reporter assay to study the effect of UBR5 on BMAL1 transcriptional activity.Finally,we used RNAi to knock down UBR5 homolog hyd in Drosophila melanogaster to explore the effect of hyd on circadian rhythm.Results:In this work,we discovered that BMAL1 interacts with E3 ubiquitin ligase UBR5 by mass spectrometric and validated this specific interaction using immunoprecipitation,immunoblotting,and immunofluorescence experiments.A series of biochemical experiments revealed that UBR5 expression reduces BMAL1 protein levels by promoting its ubiquitination and degradation.Dual-luciferase reporter assay demonstrated that UBR5 reduced BMAL1 transcriptional activity and the expression of BMAL1 downstream gene,Rev-Erba.We further showed that genetic knockdown of hyd(mammalian homolog of UBR5 in D.melanogaster)shortened circadian period.Conclusions:We discovered that BMAL1 interacted with UBR5 by mass spectrometric and validated this interaction using immunoprecipitation.UBR5 promotes BMAL1 ubiquitinantion,reduces its expression level and stability,and then atttenuates BMAL1 transcription activity and modulates circadian period.Our results discovered a ubiquitin ligase UBR5 that regulates BMAL1 stability and circadian period and elucidated the underlying molecular mechanism.