Circadian ribosome profiling reveals a role for the Period2 upstream open reading frame in sleep

Abstract

Many mammalian proteins have circadian cycles of production and degradation, and many of these rhythms are altered posttranscriptionally. We used ribosome profiling to examine posttranscriptional 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 Period2 (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 the Per2 uORF increased luciferase and fluorescence reporter expression in 3T3 cells and increased luciferase expression in PER2:LUC MEF cells. Mutation of the Per2 uORF in mice increased Per2 mRNA expression, enhanced ribosome binding on Per2 , and reduced total sleep time compared to that in wild-type mice. These results suggest that uORFs affect mRNA posttranscriptionally, which can impact physiological rhythms and sleep.

Upstream open reading frames dynamically modulate CLOCK protein translation to regulate circadian rhythm and sleep

“…Although Neurospora and Arabidopsis share similar design principles in circadian rhythms as humans and flies, the proteins involved differ significantly in both structure and conservation 35–37 . Recent studies have shown that uORFs in the mouse core clock gene Period2 ( Per2 ) play a role in temperature entrainment of the circadian clock 38 and in regulating sleep 39 . However, no significant differences in the period and amplitude of circadian rhythms were observed between wild-type and Per2 uORF mutant mice 39 .…”

“…Recent studies have shown that uORFs in the mouse core clock gene Period2 ( Per2 ) play a role in temperature entrainment of the circadian clock 38 and in regulating sleep 39 . However, no significant differences in the period and amplitude of circadian rhythms were observed between wild-type and Per2 uORF mutant mice 39 . In addition, extensive ribosomal binding to uORFs in rhythmically expressed mRNAs has been observed in mammalian cells, yet the functional impacts of these uORFs in circadian regulation have not been characterized 40,41 .…”