Circadian ribosome profiling reveals a role for thePeriod2upstream opening reading frame in sleep

Abstract

AbstractMany mammalian proteins have circadian cycles of production and degradation, and many of these rhythms are altered post-transcriptionally. We used ribosome profiling to examine post-transcriptional control of circadian rhythms by quantifying RNA translation in the liver over a 24-h period from circadian-entrained mice transferred to constant darkness conditions and by comparing ribosome binding levels to protein levels for 16 circadian proteins. We observed large differences in ribosome binding levels compared to protein levels, and we observed delays between peak ribosome binding and peak protein abundance. We found extensive binding of ribosomes to upstream open reading frames (uORFs) in circadian mRNAs, including the core clock genePeriod2 (Per2). An increase in the number of uORFs in the 5’UTR was associated with a decrease in ribosome binding in the main coding sequence and a reduction in expression of synthetic reporter constructs. Mutation of thePer2uORF increased luciferase and fluorescence reporter expression in 3T3 cells without altering the phase or period and increased luciferase expression in PER2:LUC MEF cells. Mutation of thePer2uORF in mice increased PER2 protein levels and reduced total sleep time compared to that in wild-type mice. These results suggest that post-transcriptional processes shape translation of mRNA transcripts, which can impact physiological rhythms and sleep.Significance StatementPeriod (Per)is an iconic gene in the field of circadian rhythms since its discovery in 1971 by Seymour Benzer and Ronald Konopka in fruit flies. The inhibitory feedback loop of PER protein drives circadian rhythms, and here, we add to our understanding of PER protein production by analyzing a previously unknown upstream open reading frame (uORF) in the 5’ untranslated region ofPeriod2mRNA. Mutation of thePer2uORF altered the amplitude, but not the phase or period, ofPer2expression in well-characterized cell culture models, and we observed thatPer2uORF mutant mice exhibited sleep loss, particularly during light-to-dark and dark-to-light transitions, which suggests a role for uORFs in modulating molecular and physiological circadian rhythms.