Circadian ribosome profiling reveals a role for the Period2 upstream opening reading frame in sleep.
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE231820 | GEO | 2023/09/13
REPOSITORIES: GEO
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Project description: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.
2023-05-05 | GSE201732 | GEO
Project description:Disrupted sleep-wake and molecular circadian rhythms are a feature of aging associated with metabolic disease and reduced levels of NAD+, yet whether changes in nucleotide metabolism control circadian behavioral and genomic rhythms remains unknown. Here we reveal that supplementation with the NAD+ precursor nicotinamide riboside (NR) markedly reprograms metabolic and stress-response pathways that decline with aging through inhibition of the clock repressor PER2. NR enhances BMAL1 chromatin binding genome-wide through PER2K680 deacetylation, which in turn primes PER2 phosphorylation within a domain that controls nuclear transport and stability and which is mutated in human advanced sleep phase syndrome. In old mice, dampened BMAL1 chromatin binding, transcriptional oscillations, mitochondrial respiration rhythms, and late evening activity are restored by NAD+ repletion to youthful levels with NR. These results reveal effects of NAD+ on metabolism and the circadian system with aging through the spatiotemporal control of the molecular clock.
2020-05-04 | GSE133989 | GEO
Project description:Mammalian gene expression displays widespread circadian oscillations. Rhythmic transcription underlies the core clock mechanism, but it cannot explain numerous observations made at the level of protein rhythmicity. We have used ribosome profiling in mouse liver to measure the translation of mRNAs into protein around-the-clock and at high temporal and nucleotide resolution. Transcriptome-wide, we discovered extensive rhythms in ribosome occupancy, and identified a core set of ≈150 mRNAs subject to particularly robust daily changes in translation efficiency. Cycling proteins produced from non-oscillating transcripts revealed thus far unknown rhythmic regulation associated with specific pathways (notably in iron metabolism, through the rhythmic translation of transcripts containing iron responsive elements), and indicated feedback to the rhythmic transcriptome through novel rhythmic transcription factors. Moreover, estimates of relative levels of core clock protein biosynthesis that we deduced from the data explained known features of the circadian clock better than did mRNA expression alone. Finally, we identified uORF translation as a novel regulatory mechanism within the clock circuitry. Consistent with the occurrence of translated uORFs in several core clock transcripts, loss-of-function of Denr, a known regulator of re-initiation after uORF usage and of ribosome recycling, led to circadian period shortening in cells. In summary, our data offer a framework for understanding the dynamics of translational regulation, circadian gene expression, and metabolic control in a solid mammalian organ.
2015-10-14 | GSE67305 | GEO
Project description:The peripheral circadian oscillator plays an essential role in synchronizing local physiology to operate in a circadian manner via regulation of the expression of clock-controlled genes. In the murine uterus, the endometrial stromal cells undergo proliferation and differentiation into decidual cells in response to ovarian steroids and blastocyst implantation at the early stage of pregnancy. The circadian clock genes are attenuated in the decidualizing cells only 2 days after implantation. The present study aimed to evaluate the circadian rhythms of clock genes and clock-controlled genes expressed in the rat uterus endometrial stromal cells (UESCs) during the stage of implantation. The real-time monitoring system of Per2 promoter activity was employed to precisely evaluate the generation of circadian rhythms in the UESCs prepared from transgenic rats constructed with mouse Per2 promoter-destabilized luciferase reporter gene (Per2-dLuc). During monitoring Per2-dLuc oscillation after synchronization with dexamethasone, total RNA was isolated from the cultured UESCs at four-time points (6-h interval) in the first to second phases and cDNA was synthesized. cRNA was synthesized from the double strand cDNA and hybridized on a DNA microarray. RT-qPCR was performed to confirm the expression of core clock genes revealed by DNA microarray analysis. Several clock genes such as Bmal1, Rev-erbα, and Per2 displayed significant rhythms. Of 12,252 genes showing significantly expression, 7,235 genes displayed significant alterations (p < 0.05). These genes were related to growth factors, transcription factors, receptors, channels, and enzymes. Some candidates as clock-controlled genes were evaluated by using RNA interference to Bmal1 mRNA. Down-regulation of Igf1 gene expression was observed by Bmal1 silencing, whereas the expression of Inhβa, Fas, and Caspase3 were significantly increased. These results indicate that clock-controlled genes are up- or down-regulated in rat UESCs during the stage of decidualization. DNA microarray analysis coupled with RNA interference will be helpful to understand the physiological roles of some oscillating genes in blastocyst implantation and placenta formation. The circadian clock positively or negatively regulates the expression of clock-controlled genes, including growth factors and apoptosis-related factors. To search the clock-controlled genes expressed during the period of implantation, we analyzed the clock genes and clock-controlled genes expressed in cultured uterus endometrial stromal cells prepared from pregnant rats at the stage of implantation using DNA microarray technology. We used transgenic rats constructed with mouse Per2 promoter-destabilized luciferase (Per2-dLuc) reporter gene to precisely adjust the time of gene expression. In addition, several genes of significantly expressed genes including growth factor genes and apoptosis-related genes were analyzed using RNA interference to Bmal1 mRNA whether these were controlled under circadian clockwork.
2013-07-23 | E-GEOD-49080 | biostudies-arrayexpress
Project description:Though it is well established that immunological functions of CD4+ T cells are time of day-dependent, the underlying molecular mechanisms remain largely obscure. To address the question whether T cells themselves harbor a functional clock driving circadian rhythms of immune function, we analyzed clock gene expression and immune responses of CD4+ T cells purified from blood of healthy subjects at different time points throughout the day. Circadian clock function as well as immune function was further analyzed in cultivated T cells and circadian clock reporter systems. We found robust rhythms of clock gene expression as well as, after stimulation, of IFN-g production and CD40L expression in both freshly isolated and in cultured CD4+ T cells. Moreover, circadian luciferase reporter activities in CD4+ T cells and in thymic sections from PER2::LUCIFERASE reporter mice suggest that endogenous T cell clock rhythms are self-sustained under constant culture conditions. Microarray analysis of stimulated CD4+ T cell cultures revealed a rhythmic regulation of the NF-kB pathway as a candidate mechanism regulating circadian immune responses. Collectively, these data demonstrate for the first time that CD4+ T cell responses are regulated by an intrinsic cellular circadian oscillator capable of driving rhythmic adaptive immune responses in vitro and in vivo. The study is designed with 3 biological replicates from three different time points.
2012-04-30 | E-GEOD-29583 | biostudies-arrayexpress
Project description:Upstream open reading frames (uORFs) initiate translation within mRNA 5’ leaders,and have the potential to altermain coding sequence(CDS) translationontranscripts in which they reside. Ribosome profiling(RP)studies suggest that translating ribosomes are pervasive within 5’ leadersacross model systems. However, the significance of this observationremains unclear. To explore a role for uORF usage in neuronal differentiation, we performed RP on undifferentiated and differentiated human neuroblastoma cells. Using a spectral coherence algorithm (SPECtre),we identify4,954uORFsacross31%of all neuroblastoma transcripts. These uORFspredominantly utilize non-AUG initiationcodonsand exhibit translational efficiencies(TE)comparable to annotated coding regions. Usage of both AUG initiated uORFs and aconservedandconsistently translated subset of non-AUG initiated uORFs correlateswith repressed CDS translation. Ribosomal protein transcripts are enriched in uORFs, and select uORFs on such transcripts were validated for expression. Withneuronal differentiation, we observedan overall positive correlation between translational shifts in uORF/CDSpairs. However,a subset of transcripts exhibit inverse shifts in translation of uORF/CDSpairs. TheseuORFsare enriched in AUG initiation sites, non-overlapping, and shorterin length. Cumulatively, CDSs downstream of uORFs characterized by persistent translation show smaller shifts in TE withneuronal differentiation relative to CDSs without a predicted uORF, suggesting that fluctuations in CDS translation are buffered by uORF translation. In sum, this work provides insights into the dynamic relationshipsand potential regulatory functionsof uORF/CDS pairs in a model of neuronal differentiation.
2018-09-07 | GSE119615 | GEO
Project description:Circadian rhythms are a series of endogenous autonomous 24-hour oscillations generated by the circadian clock. At the molecular level, the circadian clock is generated by a transcription-translation feedback loop, where BMAL1 and CLOCK transcription factors of the positive arm activate the expression of CRYPTOCHROME and PERIOD (PER) genes of the negative arm as well as the circadian clock-regulated genes. In this project, we aimed at finding the interactome of PER2 protein in human U2OS osteosarcoma cell line using proximity-dependent biotin identification (BioID) technique. U2OS clones overexpressing PER2-BioID2 or BioID2 were treated with dexamethasone in order to reset the circadian rhythm, and cells were then incubated in biotin-containing media for 12 hours to label the proteins in close proximity of PER2-BioID2. Samples were collected after 36 and 48 hours of the resetting to identify the labeled proteins by mass spectrometry. In addition to known interactors such as CRY1 and CRY2, many novel interactors were identified. In summary, we obtained a network of PER2 interactome and confirmed some of the novel interactions using classical the co-immunoprecipitation method.
2022-04-22 | PXD025179 | Pride
Project description:Though it is well established that immunological functions of CD4+ T cells are time of day-dependent, the underlying molecular mechanisms remain largely obscure. To address the question whether T cells themselves harbor a functional clock driving circadian rhythms of immune function, we analyzed clock gene expression and immune responses of CD4+ T cells purified from blood of healthy subjects at different time points throughout the day. Circadian clock function as well as immune function was further analyzed in cultivated T cells and circadian clock reporter systems. We found robust rhythms of clock gene expression as well as, after stimulation, of IFN-g production and CD40L expression in both freshly isolated and in cultured CD4+ T cells. Moreover, circadian luciferase reporter activities in CD4+ T cells and in thymic sections from PER2::LUCIFERASE reporter mice suggest that endogenous T cell clock rhythms are self-sustained under constant culture conditions. Microarray analysis of stimulated CD4+ T cell cultures revealed a rhythmic regulation of the NF-kB pathway as a candidate mechanism regulating circadian immune responses. Collectively, these data demonstrate for the first time that CD4+ T cell responses are regulated by an intrinsic cellular circadian oscillator capable of driving rhythmic adaptive immune responses in vitro and in vivo.
2012-05-01 | GSE29583 | GEO
Project description:The 5' untranslated region (UTR) sequence of eukaryotic mRNAs may contain upstream open reading frames (uORFs), which can regulate translation of the main open reading frame (mORF). The current model of translational regulation by uORFs posits that when a ribosome scans an mRNA and encounters a uORF, translation of that uORF can prevent ribosomes from reaching the mORF and cause decreased mORF translation. In this study, we first observed that rare variants in the 5' UTR dysregulate protein abundance. Upon further investigation, we found that rare variants near the start codon of uORFs can repress or derepress mORF translation, causing allelic changes in protein abundance. This finding holds for common variants as well, and common variants that modify uORF start codons also contribute disproportionately to metabolic and whole-plant phenotypes, suggesting that translational regulation by uORFs serves an adaptive function. These results provide evidence for the mechanisms by which natural sequence variation modulates gene expression, and ultimately, phenotype.
2021-11-02 | PXD026378 | Pride
Project description:Circadian rhythms are daily physiological and behavioral changes governed by an internal molecular clock, and dysfunctions in circadian rhythms have long been associated with various neurodegenerative diseases. Abnormal sleep-wake cycle often precedes the onset of cognitive and motor symptoms in patients, while the pathological changes may further exacerbate the disturbance in circadian cycle. It is unclear whether dysregulated circadian rhythm is a consequence of, or a contributing factor for, neurodegeneration. In addition, the evidence directly connecting the neurodegeneration-associated proteins to core circadian clock gene expression remains sparse. Here we show that FUS, a RNA-binding protein implicated in the pathogenesis of ALS and frontotemporal dementia, exhibits a bi-directional regulation with circadian rhythm. Our meta-analysis of RNAseq datasets and subsequent biochemical analysis revealed FUS as a gene regulated by circadian oscillation. Furthermore, NR1D1 binds the FUS promoter and regulates the amplitude of FUS oscillation. Meanwhile, FUS is recruited by transcriptional co-repressor PSF, and is found in the same complex as Bmal-Clock to repress Per2 expression. More strikingly, in cells and brain tissues from homozygous knock-in rats, the pathogenic R521C mutant FUS significantly alters the oscillation patterns of core circadian genes even at young age. Therefore, our results have revealed a novel bi-directional mechanism whereby dysregulated circadian clock and FUS expression may exacerbate neurodegeneration via mutual influence.
2017-04-12 | GSE77572 | GEO