Circadian ribosome profiling reveals a role for the<i>Period2</i>upstream open reading frame in sleep

<jats:title>Abstract</jats:title><jats:p>Many 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 gene<jats:italic>Period2 (Per2)</jats:italic>. 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 the<jats:italic>Per2</jats:italic>uORF increased luciferase and fluorescence reporter expression in 3T3 cells and increased luciferase expression in PER2:LUC MEF cells. Mutation of the<jats:italic>Per2</jats:italic>uORF in mice increased<jats:italic>Per2</jats:italic>mRNA expression, enhanced ribosome binding on<jats:italic>Per2</jats:italic>, and reduced total sleep time compared to that in wild-type mice. These results suggest that uORFs affect mRNA post-transcriptionally, which can impact physiological rhythms and sleep.</jats:p><jats:sec><jats:title>Significance Statement</jats:title><jats:p><jats:italic>Period (Per)</jats:italic>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. We show that<jats:italic>Per2</jats:italic>is regulated by an upstream open reading frame (uORF) in the 5’ untranslated region of<jats:italic>Period2</jats:italic>mRNA. Mutation of the<jats:italic>Per2</jats:italic>uORF altered the amplitude of luciferase reporter expression in well-characterized cell culture models.<jats:italic>Per2</jats:italic>uORF mutant mice had significantly elevated<jats:italic>Per2</jats:italic>mRNA levels and 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.</jats:p></jats:sec>