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1. XAP5 CIRCADIAN TIMEKEEPER regulates RNA splicing and the circadian clock by genetically separable pathways

   https://doi.org/10.1093/plphys/kiad193  

   Zhang, Hongtao; Kumimoto, Roderick W; Anver, Shajahan; Harmer, Stacey L (March 2023, Plant Physiology)

   Abstract The circadian oscillator allows organisms to synchronize their cellular and physiological activities with diurnal environmental changes. In plants, the circadian clock is primarily composed of multiple transcriptional–translational feedback loops. Regulators of post-transcriptional events, such as precursor messenger RNAs (pre-mRNA) splicing factors, are also involved in controlling the pace of the clock. However, in most cases the underlying mechanisms remain unclear. We have previously identified XAP5 CIRCADIAN TIMEKEEPER (XCT) as an Arabidopsis thaliana circadian clock regulator with uncharacterized molecular functions. Here, we report that XCT physically interacts with components of the spliceosome, including members of the Nineteen Complex (NTC). PacBio Iso-Seq data show that xct mutants have transcriptome-wide pre-mRNA splicing defects, predominantly aberrant 3′ splice site selection. Expression of a genomic copy of XCT fully rescues those splicing defects, demonstrating that functional XCT is important for splicing. Dawn-expressed genes are significantly enriched among those aberrantly spliced in xct mutants, suggesting that the splicing activity of XCT may be circadian regulated. Furthermore, we show that loss-of-function mutations in PRP19A or PRP19B, 2 homologous core NTC components, suppress the short circadian period phenotype of xct-2. However, we do not see rescue of the splicing defects of core clock genes in prp19 xct mutants. Therefore, our results suggest that XCT may regulate splicing and the clock function through genetically separable pathways. 

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2. Core circadian clock genes control molecular and behavioral circatidal rhythms in Parhyale hawaiensis

   https://doi.org/10.64898/2026.02.27.708297  

   Louis, Victoria; Bellido, Zachary; Helfenbein, Adam; Rosenthal, Joshua; Emery, Patrick (March 2026, bioRxiv)

   Abstract Marine organisms exhibit 12.4-hour rhythms of gene expression, physiology and behavior synchronized by tidal cues. The mechanism underlying these circatidal rhythms, and its overlap with the circadian clockwork, has remained elusive. However, recent studies showed that the core circadian gene BMAL1 sustains circatidal behavior in crustaceans. Therefore, we mutagenized the other three core circadian clock genes (PhCry2, PhPerandPhClk) inP. hawaiensis, a marine amphipod. We found that they are necessary for both circadian and circatidal behaviors. Moreover, all four core circadian genes are critical for 24-h oscillations of mRNA levels in circadian brain neurons and 12.4-h mRNA rhythms in circatidal neurons. Unexpectedly, the mutants indicate that PhCLK repressesPhPerexpression independently of PhBMAL1 specifically in circatidal neurons. Our study thus reveals that circadian and circatidal clocks share four core molecular components, but their transcriptional wiring differs. 

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3. The period gene alters daily and seasonal timing in Ostrinia nubilalis

   https://doi.org/10.1101/2024.11.02.621642  

   Dayton, Jacob N; Dopman, Erik B (November 2024, bioRxiv)

   Summary The timing of insects’ daily (feeding, movement) and seasonal (diapause, migration) rhythms affects their population dynamics and distribution. Yet, despite their implications for insect conservation and pest management, the genetic mechanisms underlying variation in timing are poorly understood. Prior research in the European corn borer moth (Ostrinia nubilalis) associated ecotype differences in seasonal diapause and daily activity with genetic variation at the circadian clock geneperiod(per). Here, we demonstrate that populations with divergent allele frequencies atperexhibit differences in daily behavior, seasonal development, and the expression of circadian clock genes. Specifically, later daily activity and shortened diapause were associated with a reduction and delay in the abundance of cyclingpermRNA. CRISPR/Cas9-mediated mutagenesis revealed thatperand/or an intact circadian clock network were essential for the appropriate timing of daily behavior and seasonal responsiveness. Furthermore, a reduction ofpergene dosage inperheterozygous mutants (per^-/+) pleiotropically decreased the diapause incidence, shortened post-diapause development, and delayed the timing of daily behavior, in a manner phenotypically reminiscent of wild-type individuals. Altogether, this combination of observational and experimental research strongly suggests thatperis a master regulator of biological rhythms and may contribute to the observed life cycle differences between bivoltine (two generation) and univoltine (one generation)O. nubilalis. HighlightsNatural ecotypes with divergentperiod(per) genotypes differ in their daily and seasonal responses to photoperiodLater daily activity, reduced diapause incidence, and shorter post-diapause development is associated with reducedpermRNA abundanceperis essential for short-day recognition and daily timingReducedpergene dosage shortened post-diapause development and delayed locomotor activity 

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4. Investigating the Roles for Essential Genes in the Regulation of the Circadian Clock in Synechococcus elongatus Using CRISPR Interference

   https://doi.org/10.1177/07487304241228333  

   Boodaghian, Nouneh; Park, Hyunsook; Cohen, Susan E (June 2024, Journal of Biological Rhythms)

   Circadian rhythms are found widely throughout nature where cyanobacteria are the simplest organisms, in which the molecular details of the clock have been elucidated. Circadian rhythmicity in cyanobacteria is carried out via the KaiA, KaiB, and KaiC core oscillator proteins that keep ~24 h time. A series of input and output proteins—CikA, SasA, and RpaA—regulate the clock by sensing environmental changes and timing rhythmic activities, including global rhythms of gene expression. Our previous work identified a novel set of KaiC-interacting proteins, some of which are encoded by genes that are essential for viability. To understand the relationship of these essential genes to the clock, we applied CRISPR interference (CRISPRi) which utilizes a deactivated Cas9 protein and single-guide RNA (sgRNA) to reduce the expression of target genes but not fully abolish their expression to allow for survival. Eight candidate genes were targeted, and strains were analyzed by quantitative real-time PCR (qRT-PCR) for reduction of gene expression, and rhythms of gene expression were monitored to analyze circadian phenotypes. Strains with reduced expression of SynPCC7942_0001, dnaN, which encodes for the β-clamp of the replicative DNA polymerase, or SynPCC7942_1081, which likely encodes for a KtrA homolog involved in K⁺ transport, displayed longer circadian rhythms of gene expression than the wild type. As neither of these proteins have been previously implicated in the circadian clock, these data suggest that diverse cellular processes, DNA replication and K⁺ transport, can influence the circadian clock and represent new avenues to understand clock function. 

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5. Circadian clock proteins KaiB and Rbp2 of Synechococcus elongatus display oscillations in their subcellular localization patterns

   https://doi.org/10.1128/spectrum.01845-25  

   Bevir, Harry J; Hooper, Christopher C; Saikley, Parker; Chaljian, Tanya; Lin, Yahan; Cohen, Susan E (February 2026, Microbiology Spectrum)

   Kolodkin-Gal, Ilana (Ed.)

   ABSTRACT Circadian rhythms generated by molecular clocks time a diverse array of physiological cycles in many organisms. Cyanobacteria are currently the only prokaryotic system with a robust and rigorously tested circadian clock, which is carried out by KaiA, KaiB, and KaiC core oscillator proteins. This KaiABC oscillator drives global rhythms in gene expression, compaction of the chromosome, natural transformation, and timing of cell division in the model organismSynechococcus elongatusPCC 7942. The oscillator proteins have been previously shown to undergo changes in their subcellular localization patterns, where KaiA and KaiC are diffuse throughout the cell during the day and localize to or near one pole of the cell at night. However, functional fluorescent fusions of KaiB to track subcellular localization could not be obtained. More recently, we have described the identification of Rbp2 as a member of the extended clock network that associates with KaiC in a localized state. However, the subcellular localization patterns of KaiB and Rbp2 remain uncharacterized, and the mechanism driving the dynamic localization of circadian proteins remains undescribed. Here, we describe the patterns of KaiB and Rbp2 subcellular localization over the course of the day. Specifically, we demonstrate that KaiB and Rbp2 form polar foci and co-localize with KaiC at night using both traditional fusions to fluorescent proteins and endogenously expressed antibodies. We show that the RNA-binding activity of Rbp2 is necessary for the robust formation of Rbp2 foci. We propose a model by which Rbp2 drives circadian protein localization via its RNA binding activity.IMPORTANCECircadian rhythms driven by a circadian clock are required to time a wide range of physiological and metabolic processes. In cyanobacteria, the circadian clock is required for optimal fitness in environmental cycles. Where the clock proteins are located within the cell and how this helps them to synchronize with the environment and time rhythmic behaviors is incompletely understood. Here, we demonstrate that recently identified extended clock protein Rbp2 and core oscillator protein KaiB localize to the cell poles at night and co-localize with other members of the clock complex. We also demonstrate that the RNA binding activity of Rbp2 is necessary for the polar localization of Rbp2. Moreover, the use of endogenously expressed antibodies introduces a new and robust method for imaging challenging proteins in bacteria that is applicable across various fields. 

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