A surprising way to disrupt sleep
Researchers from Osaka University, the University of Tokyo, and the Queensland Universityof Technology identified a structure in the circadian mRNA Period2 that affects the sleepwake cycle, particularly during the early morning and late evening.
Circadian rhythms, the internal biological clocks that regulate our daily activities, areessential for maintaining health and well-being. While the role of transcription in theserhythms is well-established, a new study sheds light on the critical importance of posttranscriptional processes. The research, titled “Circadian ribosome profiling reveals a role for
the Period2 upstream open reading frame in sleep,” to be published in PNAS, redefines ourunderstanding of how translation and post-transcriptional processes influence the body’s
internal clock and its impact on sleep patterns.
Timing Is Everything: By using ribosome profiling, the research team examined the timingof ribosome binding in relation to peak protein and RNA levels. They discovered significant
differences in the timing of these processes, suggesting a complex post-translational controlof circadian protein production. “We built upon our previous work to precisely quantify thelevel of circadian proteins in mice held in constant darkness over a 24-hour period, which
controls for the confounding effects of light,” says corresponding author Hiroki Ueda. “Usingribosome profiling, I wanted to see how the binding of ribosomes to RNA related to thetiming of when those proteins actually got made. And by trying to answer this basic timing
question, we found that ribosomes bind an upstream open reading frame in Period2 whichaltered the amplitude of circadian rhythms and disrupted sleep in mice,” adds lead author
Arthur Millius.
uORFs: Silent Regulators Speak Out: The researchers found many upstream open
reading frames (uORFs) in the 5’ untranslated region of circadian mRNAs, which is the partof RNA before the so-called “coding sequence” that gets translated by ribosomes intoprotein. These uORFs were associated with reduced ribosome binding in the main coding
sequence and reduced reporter expression in a variety of circadian assays tested by theresearchers suggesting a role for uORFs in shaping circadian protein expression. “Abouthalf of genes in mice and humans have at least one uORF,” explains Dimitri Perrin, theteam’s bioinformatician, “but it’s particularly interesting that about 75% of genes associatedwith circadian rhythms have an uORF, which means that circadian rhythms are particularly
susceptible to this type of post-transcriptional regulation.”
Per2 uORF Mutation and Sleep: Mutating the uORF in the core clock gene Period2 (Per2)
yielded intriguing results. It boosted Per2 mRNA expression while significantly reducing total
sleep duration in mice, particularly during transitions between light and dark. “Our sleep datasuggests that disrupting uORFs can have physiological impacts on mice behavior, whichshows that you don’t have to mutate the protein to have an effect,” explains Rikuhiro
Yamada who analyzed the phenotype of the mice using the lab’s snappy sleep stager
system. “We performed ribosome profiling on the Per2 uORF mutant mice, and althoughribosome binding was enhanced in the mutant mice, it was the increase in Per2 mRNAlevels that was the most surprising,” continues Arthur Millius. “We thought uORFs would
primarily affect mRNA translation, but it turns out they might also trigger an RNA degradation
response similar to nonsense mediated decay.”
PER2 protein is at the center of the inhibitory feedback loop that underlies the molecularmechanisms that control circadian rhythms, but its importance goes beyond just regulating
sleep. Per2 expression is deregulated in breast cancer, downregulated in patients with acute
myeloid leukemia, and disruption of Per2 results in tumorgenesis in mice. Therefore,
understanding the post-transcriptional processes that shape Per2 expression has wide1

ranging implications for the various fields, including circadian rhythms and medicine, andcould provide new insights into how cancer hijacks a cell’s normal circadian program or help
improve drugs with time-dependent therapeutic efficacy.

Fig. 1.
Disruption of the Per2 uORF disrupts the amplitude of circadian rhythms (left) and reduces
sleep in mice (right).
License: CC BY 4.0###The article, “Circadian ribosome profiling reveals a role for the Period2 upstream open
reading frame in sleep”, was published in PNAS at DOI:
https://doi.org/10.1073/pnas.2214636120.
Keywords: Circadian rhythms, Ribosome profiling, uORF, Mass spectrometry, RNA, Sleepwake cycle, Post-transcriptional processes, Period2
Journal: Proceedings of the National Academy of Sciences (PNAS) USA.
Title: Circadian ribosome profiling reveals a role for the Period2 upstream open reading
frame in sleep.
Authors: Arthur Millius, Rikuhiro Yamada, Hiroshi Fujishima, Kazuhiko Maeda, Daron M.
Standley, Kenta Sumiyama, Dimitri Perrin, and Hiroki R. Ueda
DOI: 10.1073/pnas.2214636120

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