PRD-2 directly regulates casein kinase I and counteracts nonsense-mediated decay in the Neurospora circadian clock

Abstract

Circadian clocks in fungi and animals are driven by a functionally conserved transcription–translation feedback loop. In Neurospora crassa, negative feedback is executed by a complex of Frequency (FRQ), FRQ-interacting RNA helicase (FRH), and casein kinase I (CKI), which inhibits the activity of the clock’s positive arm, the White Collar Complex (WCC). Here, we show that the prd-2 (period-2) gene, whose mutation is characterized by recessive inheritance of a long 26 hr period phenotype, encodes an RNA-binding protein that stabilizes the ck-1a transcript, resulting in CKI protein levels sufficient for normal rhythmicity. Moreover, by examining the molecular basis for the short circadian period of upf-1prd-6 mutants, we uncovered a strong influence of the Nonsense-Mediated Decay pathway on CKI levels. The finding that circadian period defects in two classically derived Neurospora clock mutants each arise from disruption of ck-1a regulation is consistent with circadian period being exquisitely sensitive to levels of casein kinase I.

A conditional Smg6 mutant mouse model reveals circadian clock regulation through the nonsense-mediated mRNA decay pathway

“…However, an opposite drive to attract and retain NMD regulation would be plausible as well-acting on endogenous transcripts, such as Cry2, whose intrinsic instability is physiologically important. These ideas are in line with findings that, in the circadian systems of Neurospora (15,16), Arabidopsis (13), and Drosophila (14), specific roles for NMD have emerged as well.…”

“…Mechanisms that have been implicated in posttranscriptionally regulating rhythmic mRNAs in mammals include microRNA (miRNA)-mediated regulation (11) and regulated deadenylation (12). With regard to a possible involvement of NMD, evidence for roles in the circadian system has been reported from fungi, plants, and flies (13)(14)(15)(16). In mammals, the involvement of NMD in eliminating erroneous, rhythmic alternative splicing products is documented (17).…”

Nutritional compensation of the circadian clock is a conserved process influenced by gene expression regulation and mRNA stability

“…"Het" indicates heterokaryon strains derived from the Neurospora whole genome deletion collection, which were maintained on hygromycin selection medium prior to the bioluminescence race tube assays (A). The CKI 3 0 UTR mutant was constructed by C-terminal epitope-tagging (V5-10xHis-approximately 1.4-fold increase in wc-2 and an approximately 1.4-fold decrease in frh mRNA expression levels (Figure 5B from [52,53]). wc-2 is up-regulated in NMD mutants, which could easily explain the under-compensation phenotype because wc-1 overexpression alone is sufficient to drive nutritional under-compensation [33].…”

“…The NMD machinery is required for circadian function through its regulation of ck-1a expression [52], and ck-1a transcript stability appears to undergo additional, nutrient-dependent regulation because deletion of the ck-1a 3 0 UTR (its NMD-targeting sequence) causes nutritional under-compensation (Fig 3C). The APA landscape in Neurospora is dynamic across nutrient conditions and requires CFIm complex activity (Fig 4).…”

A role for gene expression and mRNA stability in nutritional compensation of the circadian clock

“…Custom ImageJ Macros to quantify the growth front or old tissue regions of race tubes from Princeton *.spe image files are available at: https://github.com/cmk35. To calculate the circadian period length, background-corrected luminescence traces were run through two different algorithms and averaged as previously described (Kelliher et al 2020 eLife). Race tubes period lengths were measured using ChronOSX 2.1 software.…”