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A defining property of circadian clocks is temperature compensation, characterized by the resilience of their near 24-hour free-running periods against changes in environmental temperature within the physiological range. While temperature compensation is evolutionary conserved across different taxa of life and has been studied within many model organisms, its molecular underpinnings remain elusive. Posttranscriptional regulations such as temperature-sensitive alternative splicing or phosphorylation have been described as underlying reactions. Here, we show that knockdown of cleavage and polyadenylation specificity factor subunit 6 (CPSF6), a key regulator of 3′-end cleavage and polyadenylation, significantly alters circadian temperature compensation in human U-2 OS cells. We apply a combination of 3′-end-RNA-seq and mass spectrometry–based proteomics to globally quantify changes in 3′ UTR length as well as gene and protein expression between wild-type and CPSF6 knockdown cells and their dependency on temperature. Since changes in temperature compensation behavior should be reflected in alterations of temperature responses within one or all of the 3 regulatory layers, we statistically assess differential responses upon changes in ambient temperature between wild-type and CPSF6 knockdown cells. By this means, we reveal candidate genes underlying circadian temperature compensation, including eukaryotic translation initiation factor 2 subunit 1 (EIF2S1)., Peer Reviewed

Nonsense-mediated messenger RNA (mRNA) decay (NMD) has been intensively studied as a surveillance pathway that degrades erroneous transcripts arising from mutations or RNA processing errors. While additional roles in physiological control of mRNA stability have emerged, possible functions in mammalian physiology in vivo remain unclear. Here, we created a conditional mouse allele that allows converting the NMD effector nuclease SMG6 from wild-type to nuclease domain-mutant protein. We find that NMD down-regulation affects the function of the circadian clock, a system known to require rapid mRNA turnover. Specifically, we uncover strong lengthening of free-running circadian periods for liver and fibroblast clocks and direct NMD regulation of Cry2 mRNA, encoding a key transcriptional repressor within the rhythm-generating feedback loop. Transcriptome-wide changes in daily mRNA accumulation patterns in the entrained liver, as well as an altered response to food entrainment, expand the known scope of NMD regulation in mammalian gene expression and physiology.

Nonsense-mediated mRNA decay (NMD) has been intensively studied as a surveillance pathway that degrades erroneous transcripts arising from mutations or RNA processing errors. While additional roles in controlling regular mRNA stability have emerged, possible functions in mammalian physiology in vivo have remained unclear. Here, we report a novel conditional mouse allele that allows converting the NMD effector nuclease SMG6 from wild-type to nuclease domain-mutant protein. We analyzed how NMD downregulation affects the function of the circadian clock, a system known to require rapid mRNA turnover. We uncover strong lengthening of free-running circadian periods for liver and fibroblast clocks, and direct NMD regulation of Cry2 mRNA, encoding a key transcriptional repressor within the rhythm-generating feedback loop. In the entrained livers of Smg6 mutant animals we reveal transcriptome-wide alterations in daily mRNA accumulation patterns, altogether expanding the known scope of NMD regulation in mammalian gene expression and physiology.

The skin is the largest human organ with a circadian clock that regulates its function. Although circadian rhythms in specific functions are known, rhythms in the proximal clock output, gene expression, in human skin have not been thoroughly explored. This work reports 24 h gene expression rhythms in two skin layers, epidermis and dermis, in a cohort of young, healthy adults, who maintained natural, regular sleep-wake schedules. 10% of the expressed genes showed such diurnal rhythms at the population level, of which only a third differed between the two layers. Amplitude and phases of diurnal gene expression varied more across subjects than layers, with amplitude being more variable than phases. Expression amplitudes in the epidermis were larger and more subject-variable, while they were smaller and more consistent in the dermis. Core clock gene expression was similar across layers at the population-level, but were heterogeneous in their variability across subjects. We also identified small sets of biomarkers for internal clock phase in each layer, which consisted of layer-specific non-core clock genes. This work provides a valuable resource to advance our understanding of human skin and presents a novel methodology to quantify sources of variability in human circadian rhythms., Peer Reviewed

The circadian clock modulates key physiological processes in many organisms. This widespread role of circadian rhythms is typically characterized at the molecular level by profiling the transcriptome at multiple time points. Subsequent analysis identifies transcripts with altered rhythms between control and perturbed conditions, that is, are differentially rhythmic (DiffR). Commonly, Venn diagram analysis (VDA) compares lists of rhythmic transcripts to catalog transcripts with rhythms in both conditions, or that have gained or lost rhythms. However, unavoidable errors in rhythmicity detection propagate to the final DiffR classification resulting in overestimated DiffR. We show using artificial experiments on biological data that VDA indeed produces excessive false DiffR hits both in the presence and absence of true DiffR transcripts. We review and benchmark hypothesis testing and model selection approaches that instead compare circadian amplitude and phase of transcripts between the two conditions. These methods identify transcripts that ‘gain’, ‘lose’, ‘change’, or have the ‘same’ rhythms; the third category is missed by VDA. We reanalyzed three studies on the interplay between metabolism and the clock in the mouse liver that used VDA. We found not only fewer DiffR transcripts than originally reported, but VDA overlooked many relevant DiffR transcripts. Our analyses confirmed some and contradicted other conclusions in the original studies and also generated novel insights. Our conclusions equally apply to circadian studies using other omics technologies. We believe that avoiding Venn diagrams and using our convenient r‐package comparerhythms will improve the reliability of analyses in chronobiology., Comparing rhythms with and without an intervention reveals the functioning of the circadian system. High‐throughput studies of clock outputs (transcripts, proteins, etc.) typically compare lists of rhythmic outputs in each condition using Venn diagrams. This approach incorrectly predicts too many altered rhythms, while also overlooking some rhythm changes. Direct comparison of amplitudes and phases using R‐package comparerhythms fixes this problem and reveals limited circadian remodeling due to metabolic changes. image, Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659, Peer Reviewed