New study reveals critical role of post-transcriptional processes in circadian rhythms and sleep

Circadian rhythms, those internal biological clocks that govern our daily routines, play a pivotal role in maintaining our overall health and well-being. Although we have long recognized the significance of transcription in these rhythms, a recent study provides fresh insights into the crucial role of post-transcriptional processes.

The study, published as “Circadian ribosome profiling reveals the significance of the Period2 upstream open reading frame in regulating sleep” in PNAS, reshapes our comprehension of how translation and post-transcriptional mechanisms shape our body’s internal timekeeping and how they affect our sleep patterns.

Timing is everything

Through the utilization of ribosome profiling techniques, the research team meticulously examined the synchronization between ribosome binding and the peaks in protein and RNA levels. Their findings unveiled noteworthy disparities in the timing of these processes, indicating the existence of intricate post-translational controls over circadian protein synthesis.

Hiroki Ueda, the corresponding author, explains, “Expanding upon our prior work, we meticulously quantified the levels of circadian proteins in mice subjected to constant darkness for a full 24-hour cycle, effectively eliminating any potential influence of light.”

Lead author Arthur Millius elaborates, “My aim with ribosome profiling was to elucidate the correlation between ribosome binding to RNA and the actual protein synthesis timing. In our pursuit of this fundamental timing question, we stumbled upon the discovery that ribosomes engage an upstream open reading frame in Period2, thereby altering the amplitude of circadian rhythms and disrupting sleep patterns in mice.”

uORFs: Silent regulators speak out

The research team made a significant discovery by identifying numerous upstream open reading frames (uORFs) located in the 5′ untranslated region of circadian mRNA. This region precedes the coding sequence, the part of RNA translated by ribosomes into proteins. What’s intriguing is that these uORFs were linked to reduced ribosome binding within the primary coding sequence and diminished reporter expression across various circadian assays conducted by the researchers. This strongly suggests that uORFs play a pivotal role in molding the expression of circadian proteins.

Dimitri Perrin, the team’s bioinformatician, notes, “It’s noteworthy that approximately half of the genes in both mice and humans contain at least one uORF. However, what’s particularly fascinating is that roughly 75% of genes associated with circadian rhythms possess uORFs, implying that circadian processes are particularly susceptible to this form of post-transcriptional regulation.”

Per2 uORF mutation and sleep

Introducing mutations to the upstream open reading frame (uORF) within the core clock gene, Period2 (Per2), led to intriguing outcomes. It amplified the expression of Per2 mRNA while significantly reducing the total sleep duration in mice, especially during the transitions between light and dark periods.

Rikuhiro Yamada, responsible for analyzing the mice’s behavior using the lab’s snappy sleep stager system, comments, “Our sleep data indicates that disturbing uORFs can have substantial physiological effects on mouse behavior, highlighting that altering the protein itself isn’t necessary to observe an impact.”

Arthur Millius, during ribosome profiling on the Per2 uORF mutant mice, adds, “Surprisingly, we observed not only an enhancement in ribosome binding in the mutant mice but also a remarkable increase in Per2 mRNA levels. We initially believed uORFs primarily influenced mRNA translation, but it appears they might also trigger an RNA degradation response akin to nonsense-mediated decay.”

The PER2 protein stands at the core of the inhibitory feedback loop essential for governing circadian rhythms’ molecular mechanisms. However, its significance extends beyond sleep regulation. Per2 expression irregularities are associated with breast cancer, downregulated in acute myeloid leukemia patients, and disrupting Per2 leads to tumorigenesis in mice. Hence, comprehending the post-transcriptional processes influencing Per2 expression holds broad implications across fields, including circadian rhythms and medicine. It could offer fresh insights into how cancer exploits a cell’s natural circadian program or contribute to the development of time-dependent therapeutic drugs.

Source: Osaka University

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