Timed exercise stabilizes behavioral rhythms but not molecular programs in the brain's suprachiasmatic clock

Timed daily access to a running-wheel (scheduled voluntary exercise; SVE) synchronizes rodent circadian rhythms and promotes stable, 24h rhythms in animals with genetically targeted impairment of neuropeptide signaling (Vipr2−/− mice). Here we used RNA-seq and/or qRT-PCR to assess how this neuropeptide signaling impairment as well as SVE shapes molecular programs in the brain clock (suprachiasmatic nuclei; SCN) and peripheral tissues (liver and lung). Compared to Vipr2+/+ animals, the SCN transcriptome of Vipr2−/− mice showed extensive dysregulation which included core clock components, transcription factors, and neurochemicals. Furthermore, although SVE stabilized behavioral rhythms in these animals, the SCN transcriptome remained dysregulated. The molecular programs in the lung and liver of Vipr2−/− mice were partially intact, although their response to SVE differed to that of these peripheral tissues in the Vipr2+/+ mice. These findings highlight that SVE can correct behavioral abnormalities in circadian rhythms without causing large scale alterations to the SCN transcriptome

Two Ck1δ transcripts regulated by m6A methylation code for two antagonistic kinases in the control of the circadian clock.

Fustin, J.-M., Kojima, R., Itoh, K., Chang, H.-Y., Shiqi, Y., Zhuang, B., . . . Okamura, H. (2018). Two Ck1δ transcripts regulated by m6A methylation code for two antagonistic kinases in the control of the circadian clock. Proceedings of the National Academy of Sciences of the United States of America, 115(23), 5980-5985. doi:10.1073/pnas.172137111

Promoter-bound METTL3 maintains myeloid leukaemia by m6A-dependent translation control.

N6-methyladenosine (m6A) is an abundant internal RNA modification in both coding and non-coding RNAs that is catalysed by the METTL3-METTL14 methyltransferase complex. However, the specific role of these enzymes in cancer is still largely unknown. Here we define a pathway that is specific for METTL3 and is implicated in the maintenance of a leukaemic state. We identify METTL3 as an essential gene for growth of acute myeloid leukaemia cells in two distinct genetic screens. Downregulation of METTL3 results in cell cycle arrest, differentiation of leukaemic cells and failure to establish leukaemia in immunodeficient mice. We show that METTL3, independently of METTL14, associates with chromatin and localizes to the transcriptional start sites of active genes. The vast majority of these genes have the CAATT-box binding protein CEBPZ present at the transcriptional start site, and this is required for recruitment of METTL3 to chromatin. Promoter-bound METTL3 induces m6A modification within the coding region of the associated mRNA transcript, and enhances its translation by relieving ribosome stalling. We show that genes regulated by METTL3 in this way are necessary for acute myeloid leukaemia. Together, these data define METTL3 as a regulator of a chromatin-based pathway that is necessary for maintenance of the leukaemic state and identify this enzyme as a potential therapeutic target for acute myeloid leukaemia

Activation of AMPA Receptors in the Suprachiasmatic Nucleus Phase-Shifts the Mouse Circadian Clock In Vivo and In Vitro

The glutamatergic neurotransmission in the suprachiasmatic nucleus (SCN) plays a central role in the entrainment of the circadian rhythms to environmental light-dark cycles. Although the glutamatergic effect operating via NMDAR (N-methyl D-aspartate receptor) is well elucidated, much less is known about a role of AMPAR (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor) in circadian entrainment. Here we show that, in the mouse SCN, GluR2 and GluR4 AMPAR subtypes are abundantly expressed in the retinorecipient area. In vivo microinjection of AMPA in the SCN during the early subjective night phase-delays the behavioral rhythm. In the organotypic SCN slice culture, AMPA application induces phase-dependent phase-shifts of core-clock gene transcription rhythms. These data demonstrate that activation of AMPAR is capable of phase-shifting the circadian clock both in vivo and in vitro, and are consistent with the hypothesis that activation of AMPA receptors is a critical step in the transmission of photic information to the SCN

The m6A-methylase complex recruits TREX and regulates mRNA export

N6-methyladenosine (m6A) is the most abundant internal modification of eukaryotic mRNA. This modification has previously been shown to alter the export kinetics for mRNAs though the molecular details surrounding this phenomenon remain poorly understood. Recruitment of the TREX mRNA export complex to mRNA is driven by transcription, 5' capping and pre-mRNA splicing. Here we identify a fourth mechanism in human cells driving the association of TREX with mRNA involving the m6A methylase complex. We show that the m6A complex recruits TREX to m6A modified mRNAs and this process is essential for their efficient export. TREX also stimulates recruitment of the m6A reader protein YTHDC1 to the mRNA and the m6A complex influences the interaction of TREX with YTHDC1. Together our studies reveal a key role for TREX in the export of m6A modified mRNAs

Implication of the F-Box Protein FBXL21 in circadian pacemaker function in mammals.

In mammals, the circadian clock relies on interlocked feedback loops involving clock genes and their protein products. Post-translational modifications control intracellular trafficking, functionality and degradation of clock proteins and are keys to the functioning of the clock as recently exemplified for the F-Box protein Fbxl3. The SCFFbxl3 complex directs degradation of CRY1/2 proteins and Fbxl3 murine mutants have a slower clock. To assess whether the role of Fbxl3 is phylogenetically conserved, we investigated its function in the sheep, a diurnal ungulate. Our data show that Fbxl3 function is conserved and further reveal that its closest homologue, the F-Box protein Fbxl21, also binds to CRY1 which impairs its repressive action towards the transcriptional activators CLOCK/BMAL1. However, while Fbxl3 appears to be ubiquitously expressed, Fbxl21 expression is tissue-specific. Furthermore, and in sharp contrast with Fbxl3, Fbxl21 is highly expressed within the suprachiasmatic nuclei, site of the master clock, where it displays marked circadian oscillations apparently driven by members of the PAR-bZIP family. Finally, for both Fbxl3 and Fbxl21 we identified and functionally characterized novel splice-variants, which might reduce CRY1 proteasomal degradation dependent on cell context. Altogether, these data establish Fbxl21 as a novel circadian clock-controlled gene that plays a specific role within the mammalian circadian pacemaker