The circadian pacemaker in the suprachiasmatic nuclei (SCN) of the hypothalamus maintains phase coherence in peripheral cells through metabolic, neuronal, and humoral signaling pathways. Here, we ...investigated the role of daily body temperature fluctuations as possible systemic cues in the resetting of peripheral oscillators. Using precise temperature devices in conjunction with real-time monitoring of the bioluminescence produced by circadian luciferase reporter genes, we showed that simulated body temperature cycles of mice and even humans, with daily temperature differences of only 3°C and 1°C, respectively, could gradually synchronize circadian gene expression in cultured fibroblasts. The time required for establishing the new steady-state phase depended on the reporter gene, but after a few days, the expression of each gene oscillated with a precise phase relative to that of the temperature cycles. Smooth temperature oscillations with a very small amplitude could synchronize fibroblast clocks over a wide temperature range, and such temperature rhythms were also capable of entraining gene expression cycles to periods significantly longer or shorter than 24 h. As revealed by genetic loss-of-function experiments, heat-shock factor 1 (HSF1), but not HSF2, was required for the efficient synchronization of fibroblast oscillators to simulated body temperature cycles.
It has been assumed that the suprachiasmatic nucleus (SCN) synchronizes peripheral circadian oscillators. However, this has never been convincingly shown, since biochemical time series experiments ...are not feasible in behaviorally arrhythmic animals. By using long-term bioluminescence recording in freely moving mice, we show that the SCN is indeed required for maintaining synchrony between organs. Surprisingly, however, circadian oscillations persist in the livers of mice devoid of an SCN or oscillators in cells other than hepatocytes. Hence, similar to SCN neurons, hepatocytes can maintain phase coherence in the absence of Zeitgeber signals produced by other organs or environmental cycles.
In mammals, including humans, nearly all physiological processes are subject to daily oscillations that are governed by a circadian timing system with a complex hierarchical structure. The central ...pacemaker, residing in the suprachiasmatic nucleus (SCN) of the ventral hypothalamus, is synchronized daily by photic cues transmitted from the retina to SCN neurons via the retinohypothalamic tract. In turn, the SCN must establish phase coherence between self-sustained and cell-autonomous oscillators present in most peripheral cell types. The synchronization signals (Zeitgebers) can be controlled more or less directly by the SCN. In mice and rats, feeding-fasting rhythms, which are driven by the SCN through rest-activity cycles, are the most potent Zeitgebers for the circadian oscillators of peripheral organs. Signaling through the glucocorticoid receptor and the serum response factor also participate in the phase entrainment of peripheral clocks, and these two pathways are controlled by the SCN independently of feeding-fasting rhythms. Body temperature rhythms, governed by the SCN directly and indirectly through rest-activity cycles, are perhaps the most surprising cues for peripheral oscillators. Although the molecular makeup of circadian oscillators is nearly identical in all cells, these oscillators are used for different purposes in the SCN and in peripheral organs.
The mammalian circadian timing system consists of a master pacemaker in the suprachiasmatic nucleus (SCN) in the hypothalamus, which is thought to set the phase of slave oscillators in virtually all ...body cells. However, due to the lack of appropriate in vivo recording technologies, it has been difficult to study how the SCN synchronizes oscillators in peripheral tissues. Here we describe the real-time recording of bioluminescence emitted by hepatocytes expressing circadian luciferase reporter genes in freely moving mice. The technology employs a device dubbed RT-Biolumicorder, which consists of a cylindrical cage with reflecting conical walls that channel photons toward a photomultiplier tube. The monitoring of circadian liver gene expression revealed that hepatocyte oscillators of SCN-lesioned mice synchronized more rapidly to feeding cycles than hepatocyte clocks of intact mice. Hence, the SCN uses signaling pathways that counteract those of feeding rhythms when their phase is in conflict with its own phase.
The functional consequence of N6-methyladenosine (m6A) RNA modification is mediated by “reader” proteins of the YTH family. YTH domain-containing 2 (YTHDC2) is essential for mammalian fertility, but ...its molecular function is poorly understood. Here, we identify U-rich motifs as binding sites of YTHDC2 on 3′ UTRs of mouse testicular RNA targets. Although its YTH domain is an m6A-binder in vitro, the YTH point mutant mice are fertile. Significantly, the loss of its 3′→5′ RNA helicase activity causes mouse infertility, with the catalytic-dead mutation being dominant negative. Biochemical studies reveal that the weak helicase activity of YTHDC2 is enhanced by its interaction with the 5′→3′ exoribonuclease XRN1. Single-cell transcriptomics indicate that Ythdc2 mutant mitotic germ cells transition into meiosis but accumulate a transcriptome with mixed mitotic/meiotic identity that fail to progress further into meiosis. Finally, our demonstration that ythdc2 mutant zebrafish are infertile highlights its conserved role in animal germ cell development.
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•m6A-reading capacity of YTHDC2 is not essential for mouse fertility•YTHDC2 binds U-rich motifs in 3′ UTR of mRNAs•The essential RNA helicase activity of YTHDC2 is enhanced by XRN1•Ythdc2 mutant germ cells have a mixed mitotic-meiotic identity
Li et al. show that the m6A-reading capacity of YTHDC2 is not essential for mouse fertility, but instead it binds U-rich motifs on RNAs and depends on its essential RNA helicase activity. YTHDC2 allows fine separation of the mitotic and meiotic transcriptomes as germ cells transition into meiosis.
RNA polymerase II transcripts receive a protective 5ʹ,5ʹ-triphosphate-linked 7-methylguanosine (m7G) cap, and its removal by decapping enzymes like DCP2 is critical for initiation of RNA decay. ...Alternative RNA caps can be acquired when transcription initiation uses metabolites like nicotinamide adenine dinucleotide (NAD), generating NAD-RNAs. Here, we identify human NUDT12 as a cytosolic NAD-RNA decapping enzyme. NUDT12 is active only as homodimers, with each monomer contributing to creation of the two functional catalytic pockets. We identify an ∼600-kDa dodecamer complex between bleomycin hydrolase (BLMH) and NUDT12, with BLMH being required for localization of NUDT12 to a few discrete cytoplasmic granules that are distinct from P-bodies. Both proteins downregulate gene expression when artificially tethered to a reporter RNA in vivo. Furthermore, loss of Nudt12 results in a significant upregulation of circadian clock transcripts in mouse liver. Overall, our study points to a physiological role for NUDT12 in the cytosolic surveillance of NAD-RNAs.
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•Human NUDT12 is a decapping enzyme for NAD-capped RNAs•Homodimerization of NUDT12 creates two interdependent catalytic pockets•Interaction with BLMH facilitates localization of NUDT12 to cytoplasmic granules•Nudt12 knockout mice upregulate circadian clock transcripts
Wu et al. identify NUDT12 as a decapping enzyme for NAD-capped RNAs and demonstrate a role for the enzyme in regulation of circadian clock transcripts in mice. They reveal the existence of a BLMH-NUDT12 complex that facilitates localization of NUDT12 to distinct cytoplasmic granules.
DBP (albumin D-site-binding protein), HLF (hepatic leukemia factor), and TEF (thyrotroph embryonic factor) are the three members of the PAR bZip (proline and acidic amino acid-rich basic leucine ...zipper) transcription factor family. All three of these transcriptional regulatory proteins accumulate with robust circadian rhythms in tissues with high amplitudes of clock gene expression, such as the suprachiasmatic nucleus (SCN) and the liver. However, they are expressed at nearly invariable levels in most brain regions, in which clock gene expression only cycles with low amplitude. Here we show that mice deficient for all three PAR bZip proteins are highly susceptible to generalized spontaneous and audiogenic epilepsies that frequently are lethal. Transcriptome profiling revealed pyridoxal kinase (Pdxk) as a target gene of PAR bZip proteins in both liver and brain. Pyridoxal kinase converts vitamin B6 derivatives into pyridoxal phosphate (PLP), the coenzyme of many enzymes involved in amino acid and neurotransmitter metabolism. PAR bZip-deficient mice show decreased brain levels of PLP, serotonin, and dopamine, and such changes have previously been reported to cause epilepsies in other systems. Hence, the expression of some clock-controlled genes, such as Pdxk, may have to remain within narrow limits in the brain. This could explain why the circadian oscillator has evolved to generate only low-amplitude cycles in most brain regions.
In mammals, many aspects of metabolism are under circadian control. At least in part, this regulation is achieved by core-clock or clock-controlled transcription factors whose abundance and/or ...activity oscillate during the day. The clock-controlled proline-and acidic amino acid-rich domain basic leucine zipper proteins D-site-binding protein, thyrotroph embryonic factor, and hepatic leukemia factor have previously been shown to participate in the circadian control of xenobiotic detoxification in liver and other peripheral organs. Here we present genetic and biochemical evidence that the three proline- and acidic amino acid-rich basic leucine zipper proteins also play a key role in circadian lipid metabolism by influencing the rhythmic expression and activity of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα). Our results suggest that, in liver, D-site-binding protein, hepatic leukemia factor, and thyrotroph embryonic factor contribute to the circadian transcription of genes specifying acyl-CoA thioesterases, leading to a cyclic release of fatty acids from thioesters. In turn, the fatty acids act as ligands for PPARα, and the activated PPARα receptor then stimulates the transcription of genes encoding proteins involved in the uptake and/or metabolism of lipids, cholesterol, and glucose metabolism.
Internal modification of RNAs with N6-methyladenosine (m6A) is a highly conserved means of gene expression control. While the METTL3/METTL14 heterodimer adds this mark on thousands of transcripts in ...a single-stranded context, the substrate requirements and physiological roles of the second m6A writer METTL16 remain unknown. Here we describe the crystal structure of human METTL16 to reveal a methyltransferase domain furnished with an extra N-terminal module, which together form a deep-cut groove that is essential for RNA binding. When presented with a random pool of RNAs, METTL16 selects for methylation-structured RNAs where the critical adenosine is present in a bulge. Mouse 16-cell embryos lacking Mettl16 display reduced mRNA levels of its methylation target, the SAM synthetase Mat2a. The consequence is massive transcriptome dysregulation in ∼64-cell blastocysts that are unfit for further development. This highlights the role of an m6A RNA methyltransferase in facilitating early development via regulation of SAM availability.
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•Structure of the METTL16 m6A writer domain with a unique N-terminal module•N-terminal module of METTL16 is essential for charge-based binding to RNA•METTL16 preferentially methylates adenosines within structured RNAs•Regulation of Mat2a mRNA by Mettl16 is essential for mouse embryonic development
Mendel et al. reveal the structural basis for structured RNA recognition by the mammalian m6A writer METTL16 and demonstrate its essential role in mouse early embryonic development via regulation of the SAM synthetase Mat2a mRNA.
Internal modification of RNAs with N-6-methyladenosine (m(6)A) is a highly conserved means of gene expression control. While the METTL3/METTL14 heterodimer adds this mark on thousands of transcripts ...in a single-stranded context, the substrate requirements and physiological roles of the second m(6)A writer METTL16 remain unknown. Here we describe the crystal structure of human METTL16 to reveal a methyltransferase domain furnished with an extra N-terminal module, which together form a deep-cut groove that is essential for RNA binding. When presented with a random pool of RNAs, METTL16 selects for methylation-structured RNAs where the critical adenosine is present in a bulge. Mouse 16-cell embryos lacking MeTTL16 display reduced mRNA levels of its methylation target, the SAM synthetase Mat2a. The consequence is massive transcriptome dysregulation in similar to 64-cell blastocysts that are unfit for further development. This highlights the role of an m(6)A RNA methyltransferase in facilitating early development via regulation of SAM availability.
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