The Mechanism Of RNA Methytransferase MRM1 In Neurospora Circadian Clock

To adapt to the alternation of day and night caused by the earth's self-rotation,an internal timing system,referred as“circadian clock”,is developed during the long-term evolution in almost all the living organisms.Dysregulation of the circadian clock may cause many diseases,such as sleep disorder,inflammatory bowel disease,cardiovascular disease,and cancer.It has been shown that the core oscillator of eukaryotic circadian clock is composed of transcription-translation based feedback loop.In Neurospora,transcription activator White Collar-1(WC-1)and White Collar-2(WC-2)serve as positive elements in the feedback loop,whereas clock protein FREQUENCY(FRQ)and its interacting protein FRH are negative elements.Studies in mouse have shown that m⁶A exists in mRNAs of clock genes and knock-down of METTL3 results in lengthened clock period.However,it is not clear whether m⁶A or other mRNA modification are involved in the regulation of circadian clock in Neurospora crassa.To answer this question,we first showed that there was no m⁶A on the mRNA of Neurospora crassa,but 2?-O-methylation existed.In Neurospora crassa,RNA methyltransferase MRM1 was localized both in mitochondira and cytoplasm.In mitochondria,MRM1 directly catalyzed the formation of 2?-O-methylation of Gm2453 in mitochondrial 23S rRNA.In cytoplasm,MRM1 was involved in the formation of 2?-O-methylation of cytoplasmic mRNAs.It has been reported that 2?-O-methylation inhibits the translation of cytoplasmic mRNAs.Consistent with this idea,deletion of MRM1 in Neurospora resulted in increased translation of cytoplasmic mRNAs.More importantly,our data for the first time showed that MRM1 is a novel regulator of circadian clock in Neurospora.Phenotypical and molecular results showed that deletion of MRM1 led to short period and phase advance of Neurospora circadian clock.In mrm1^KO mutant,the translation of frq mRNA was increased and resulted elevated FRQ protein levels.Importantly,deletion of MRM1 alterred the stability of frq mRNA and the recruitment of WC complex.We proposed that:on one hand,deletion of MRM1 resulted in increased frq mRNA degradation,which in turn led to short period;on the other hand,deletion of MRM1 led to early recruitment of WC complex to the promoter of frq,and thus resulted in phase advance.In this study,we identify a novel regulator of Neurospora circadian clock–RNA methyltransferase MRM1.This study will not only enhance our understanding about the biological function of 2?-O-methylation modification but also the role of post-transcriptional regulation in circadian clock.