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

ABSTRACT: Many mammalian proteins have circadian cycles of production and degradation, but de novo transcription is only responsible for a small fraction of this rhythmicity. We used ribosome profiling to quantify RNA translation in liver from circadian-entrained mice transferred to constant darkness conditions over a 24-h period and compared translation levels to absolute protein production for 16 circadian proteins. We observed a delay between translation and peak protein levels for several circadian genes covering a wide range of translation efficiencies. We found extensive binding of ribosomes to upstream open reading frames (uORFs) in circadian mRNAs, including the core clock gene Period2 (Per2). Increased uORFs in 5' UTRs was associated with decreased ribosome binding in downstream ORFs and reduced expression of synthetic reporter constructs in vitro and in single cells. Mutation of the Per2 uORF increased luciferase and fluorescence reporter expression in 3T3 cells without altering phase or period. Genomic Per2 uORF mutation using CRISPR/Cas9 increased PER2 expression in PER2:Luc MEFs and reduced sleep in Per2 uORF mutant mice. These results suggest that post-transcriptional processes shape translation of mRNA transcripts, which can impact physiological rhythms and sleep.

ORGANISM(S): Mus musculus

PROVIDER: GSE201732 | GEO | 2023/05/05

REPOSITORIES: GEO

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Project description:Translation of an mRNA in eukaryotes starts at AUG in most cases. Near-cognate codons (NCCs) such as UUG, ACG and AUU are also used as start sites at low levels in S. cerevisiae. Initiation from NCCs or AUGs in the 5’-untranslated regions (UTRs) of mRNAs can lead to translation of upstream open reading frames (uORFs) that might regulate expression of the main ORF (mORF). Although there is some circumstantial evidence that the translation of uORFs can be affected by environmental conditions, little is known about how it is affected by changes in growth temperature. Using reporter assays, we found that changes in growth temperature can affect translation from NCC start sites in yeast cells, suggesting the possibility that gene expression could be regulated by temperature by altering use of different uORF start codons. Using ribosome profiling, we provide evidence that growth temperature regulates the efficiency of translation of nearly 200 uORFs in S. cerevisiae. Of these uORFs, most that start with an AUG codon have increased translational efficiency at 37 ˚C relative to 30 ˚C and decreased efficiency at 20 ˚C. For translationally regulated uORFs starting with NCCs, we did not observe a general trend for the direction of regulation as a function of temperature, suggesting mRNA-specific features can determine the mode of temperature-dependent regulation. Consistent with this conclusion, the position of the uORFs in the 5’-leader relative to the 5’-cap and the start codon of the main ORF correlates with the direction of temperature-dependent regulation of uORF translation. We have identified several novel cases in which changes in uORF translation are inversely correlated with changes in the translational efficiency of the downstream main ORF. Our data suggest that translation of these mRNAs is subject to temperature-dependent, uORF-mediated regulation. Overall, our data suggest that alterations in the translation of specific uORFs by temperature can regulate gene expression in S. cerevisiae.

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

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