Neurospora crassa has been for decades a principal model for filamentous fungal genetics and physiology as well as for understanding the mechanism of circadian clocks. Eukaryotic fungal and animal clocks comprise transcription-translation-based feedback loops that control rhythmic transcription of a substantial fraction of these transcriptomes, yielding the changes in protein abundance that mediate circadian regulation of physiology and metabolism: Understanding circadian control of gene expression is key to understanding eukaryotic, including fungal, physiology. Indeed, the isolation of clock-controlled genes (ccgs) was pioneered in Neurospora where circadian output begins with binding of the core circadian transcription factor WCC to a subset of ccg promoters, including those of many transcription factors. High temporal resolution (2-h) sampling over 48 h using RNA sequencing (RNA-Seq) identified circadianly expressed genes in Neurospora, revealing that from ∼10% to as much 40% of the transcriptome can be expressed under circadian control. Functional classifications of these genes revealed strong enrichment in pathways involving metabolism, protein synthesis, and stress responses; in broad terms, daytime metabolic potential favors catabolism, energy production, and precursor assembly, whereas night activities favor biosynthesis of cellular components and growth. Discriminative regular expression motif elicitation (DREME) identified key promoter motifs highly correlated with the temporal regulation of ccgs. Correlations between ccg abundance from RNA-Seq, the degree of ccg-promoter activation as reported by ccg-promoter-luciferase fusions, and binding of WCC as measured by ChIP-Seq, are not strong. Therefore, although circadian activation is critical to ccg rhythmicity, posttranscriptional regulation plays a major role in determining rhythmicity at the mRNA level.A well-recognized model for fungal genetics, photobiology, and circadian systems, Neurospora is the established model for nearly all aspects of growth and metabolism among the filamentous fungi. The core clock of Neurospora crassa has been well studied; its oscillator comprises a transcription-translation feedback loop involving a complex of five core proteins, White Collar 1 (WC-1), White Collar 2 (WC-2), Frequency (FRQ), Frequency Interacting RNA Helicase (FRH), and Casein Kinase 1 (CK1), as well as several ancillary factors. Transcription factors (TFs) WC-1 and WC-2 form the white collar complex (WCC), which drives the rhythmic expression of FRQ. FRQ binds to FRH to form the FRQ/FRH complex (FFC), which then acts with CK1 on the WCC to inhibit its activity, thus closing the loop. Throughout the circadian cycle, FRQ interacts with many partners that affect the number and location of its posttranslational modifications as well as its stability; it is these posttranslational modifications of FRQ that set the length of the circadian period (1-3).Although historically much interest has focused on clock mechanism, it is through circadian control of transc...

Genome biology approaches have made enormous contributions to our understanding of biological rhythms, particularly in identifying outputs of the clock, including RNAs, proteins, and metabolites, whose abundance oscillates throughout the day. These methods hold significant promise for future discovery, particularly when combined with computational modeling. However, genome-scale experiments are costly and laborious, yielding “big data” that are conceptually and statistically difficult to analyze. There is no obvious consensus regarding design or analysis. Here we discuss the relevant technical considerations to generate reproducible, statistically sound, and broadly useful genome-scale data. Rather than suggest a set of rigid rules, we aim to codify principles by which investigators, reviewers, and readers of the primary literature can evaluate the suitability of different experimental designs for measuring different aspects of biological rhythms. We introduce CircaInSilico, a web-based application for generating synthetic genome biology data to benchmark statistical methods for studying biological rhythms. Finally, we discuss several unmet analytical needs, including applications to clinical medicine, and suggest productive avenues to address them.

Summary Protein conformation dictates a great deal of protein function. A class of naturally unstructured proteins, termed Intrinsically Disordered Proteins (IDPs), demonstrates that flexibility in structure can be as important mechanistically as rigid structure. At the core of the circadian transcription/translation feedback loop in Neurospora crassa is the protein Frequency (FRQ), shown here shown to share many characteristics of IDPs. FRQ in turn binds to Frequency Interacting RNA Helicase (FRH), whose clock function has been assumed to relate to its predicted helicase function. However, mutational analyses reveal that the helicase function of FRH is not essential for the clock, and a region of FRH distinct from the helicase region is essential for stabilizing FRQ against rapid degradation via pathway distinct from its typical ubiquitin-mediated turnover. These data lead to the hypothesis that FRQ is an IDP and that FRH acts nonenzymatically, stabilizing FRQ to enable proper clock circuitry/function.

SUMMARY Transcriptional and translational feedback loops in fungi and animals drive circadian rhythms in transcript levels that provide output from the clock, but post-transcriptional mechanisms also contribute. To determine the extent and underlying source of this regulation, we applied newly developed analytical tools to a long-duration, deeply sampled, circadian proteomics time course comprising half of the proteome. We found a quarter of expressed proteins are clock regulated, but >40% of these do not arise from clock-regulated transcripts, and our analysis predicts that these protein rhythms arise from oscillations in translational rates. Our data highlighted the impact of the clock on metabolic regulation, with central carbon metabolism reflecting both transcriptional and post-transcriptional control and opposing metabolic pathways showing peak activities at different times of day. The transcription factor CSP-1 plays a role in this metabolic regulation, contributing to the rhythmicity and phase of clock-regulated proteins.

From cyanobacteria to mammals, organisms have evolved timing mechanisms to adapt to environmental changes in order to optimize survival and improve fitness. To anticipate these regular daily cycles, many organisms manifest near 24-h cell-autonomous oscillations that are sustained by transcription–translation-based or post-transcriptional negative feedback loops that control a wide range of biological processes. With an eye to identifying emerging common themes among cyanobacterial, fungal and animal clocks, some major recent developments in the understanding of the mechanisms that regulate these oscillators and their output are discussed. These include roles for antisense transcription, intrinsically disordered proteins, codon bias in clock genes, and a more focused discussion of post-transcriptional and translational regulation as a part of both the oscillator and output.

Most pathogenic Proteus species are primarily associated with urinary tract infections, especially in persons with indwelling catheters or functional/anatomic abnormalities of the urinary tract. Urinary tract infections caused by Proteus vulgaris typically form biofilms and are resistant to commonly used antibiotics. The Rts1 conjugative plasmid from a clinical isolate of P. vulgaris carries over 300 predicted open reading frames, including antibiotic resistance genes. The maintenance of the Rts1 plasmid is ensured in part by the HigBA toxin-antitoxin system. We determined the precise mechanism of action of the HigB toxin in vivo, which is distinct from other known toxins. We demonstrate that HigB is an endoribonuclease whose enzymatic activity is dependent on association with ribosomes through the 50 S subunit. Using primer extension analysis of several test mRNAs, we showed that HigB cleaved extensively across the entire length of coding regions only at specific recognition sequences. HigB mediated cleavage of 100% of both in-frame and out-of-frame AAA sequences. In addition, HigB cleaved ϳ20% of AA sequences in coding regions and occasionally cut single As. Remarkably, the cleavage specificity of HigB coincided with one of the most frequently used codons in the ATrich Proteus spp., AAA (lysine). Therefore, the HigB-mediated plasmid maintenance system for the Rts1 plasmid highlights the intimate relationship between host cells and extrachromosomal DNA that enables the dynamic acquisition of genes that impart a spectrum of survival advantages, including those encoding multidrug resistance and virulence factors.

Our core timekeeping mechanism, the circadian clock, regulates an astonishing amount of cellular physiology and behavior, playing a vital role in organismal fitness. While the mechanics of circadian control over cellular regulation can in part be explained by the transcriptional activation stemming from the positive arm of the clock's transcription-translation negative feedback loop, research has shown that extensive circadian regulation occurs beyond transcriptional activation in fungal species and data suggest that this post-transcriptional regulation may also be preserved in mammals. To determine the extent to which circadian output is regulated post-transcriptionally in mammalian cells, we comprehensively profiled the transcriptome and proteome of murine bone marrow-derived macrophages in a high resolution, sample rich time course. We found that only 15% of the circadian proteome had corresponding oscillating mRNA and this regulation was cell intrinsic. Ontological analysis of oscillating proteins revealed robust temporal enrichment for protein degradation and translation, providing potential insights into the source of this extensive posttranscriptional regulation. We noted post-transcriptional temporal-gating across a number of connected metabolic pathways. This temporal metabolic regulation further corresponded with rhythms we observed in ATP production, mitochondrial morphology, and phagocytosis. With the strong interconnection between cellular metabolic states and macrophage phenotypes/responses, our work demonstrates that post-transcriptional circadian regulation in macrophages is broadly utilized as a tool to confer time-dependent immune function and responses. As macrophages coordinate many immunological and inflammatory functions, an understanding of this regulation provides a framework to determine the impact of circadian regulation on a wide array of disease pathologies.

Summary Background Antiplatelet therapy reduces the risk of major vascular events for people with occlusive vascular disease, although it might increase the risk of intracranial haemorrhage. Patients surviving the commonest subtype of intracranial haemorrhage, intracerebral haemorrhage, are at risk of both haemorrhagic and occlusive vascular events, but whether antiplatelet therapy can be used safely is unclear. We aimed to estimate the relative and absolute effects of antiplatelet therapy on recurrent intracerebral haemorrhage and whether this risk might exceed any reduction of occlusive vascular events. Methods The REstart or STop Antithrombotics Randomised Trial (RESTART) was a prospective, randomised, open-label, blinded endpoint, parallel-group trial at 122 hospitals in the UK. We recruited adults (≥18 years) who were taking antithrombotic (antiplatelet or anticoagulant) therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage, discontinued antithrombotic therapy, and survived for 24 h. Computerised randomisation incorporating minimisation allocated participants (1:1) to start or avoid antiplatelet therapy. We followed participants for the primary outcome (recurrent symptomatic intracerebral haemorrhage) for up to 5 years. We analysed data from all randomised participants using Cox proportional hazards regression, adjusted for minimisation covariates. This trial is registered with ISRCTN (number ISRCTN71907627). Findings Between May 22, 2013, and May 31, 2018, 537 participants were recruited a median of 76 days (IQR 29–146) after intracerebral haemorrhage onset: 268 were assigned to start and 269 (one withdrew) to avoid antiplatelet therapy. Participants were followed for a median of 2·0 years (IQR [1·0– 3·0]; completeness 99·3%). 12 (4%) of 268 participants allocated to antiplatelet therapy had recurrence of intracerebral haemorrhage compared with 23 (9%) of 268 participants allocated to avoid antiplatelet therapy (adjusted hazard ratio 0·51 [95% CI 0·25–1·03]; p=0·060). 18 (7%) participants allocated to antiplatelet therapy experienced major haemorrhagic events compared with 25 (9%) participants allocated to avoid antiplatelet therapy (0·71 [0·39–1·30]; p=0·27), and 39 [15%] participants allocated to antiplatelet therapy had major occlusive vascular events compared with 38 [14%] allocated to avoid antiplatelet therapy (1·02 [0·65–1·60]; p=0·92). Interpretation These results exclude all but a very modest increase in the risk of recurrent intracerebral haemorrhage with antiplatelet therapy for patients on antithrombotic therapy for the prevention of occlusive vascular disease when they developed intracerebral haemorrhage. The risk of recurrent intracerebral haemorrhage is probably too small to exceed the established benefits of antiplatelet therapy for secondary prevention. Funding British Heart Foundation.