A circadian clock translational control mechanism targets specific mRNAs to cytoplasmic messenger ribonucleoprotein granules.

Phosphorylation of Neurospora crassa eukaryotic initiation factor 2 α (eIF2α), a conserved translation initiation factor, is clock controlled. To determine the impact of rhythmic eIF2α phosphorylation on translation, we performed temporal ribosome profiling and RNA sequencing (RNA-seq) in wild-type (WT), clock mutant Δfrq, eIF2α kinase mutant Δcpc-3, and constitutively active cpc-3c cells. About 14% of mRNAs are rhythmically translated in WT cells, and translation rhythms for ∼30% of these mRNAs, which we named circadian translation-initiation-controlled genes (cTICs), are dependent on the clock and CPC-3. Most cTICs are expressed from arrhythmic mRNAs and contain a P-body (PB) localization motif in their 5' leader sequence. Deletion of SNR-1, a component of cytoplasmic messenger ribonucleoprotein granules (cmRNPgs) that include PBs and stress granules (SGs), and the PB motif on one of the cTIC mRNAs, zip-1, significantly alters zip-1 rhythmic translation. These results reveal that the clock regulates rhythmic translation of specific mRNAs through rhythmic eIF2α activity and cmRNPg metabolism.

Circadian Clock Control of Translation Initiation Factor eIF2α Activity Requires eIF2γ-Dependent Recruitment of Rhythmic PPP-1 Phosphatase in Neurospora crassa.

The circadian clock controls the phosphorylation and activity of eukaryotic translation initiation factor 2α (eIF2α). In Neurospora crassa, the clock drives a daytime peak in the activity of the eIF2α kinase CPC-3, the homolog of yeast and mammalian GCN2 kinase. This leads to increased levels of phosphorylated eIF2α (P-eIF2α) and reduced mRNA translation initiation during the day. We hypothesized that rhythmic eIF2α activity also requires dephosphorylation of P-eIF2α at night by phosphatases. In support of this hypothesis, we show that mutation of N. crassa PPP-1, a homolog of the yeast eIF2α phosphatase GLC7, leads to high and arrhythmic P-eIF2α levels, while maintaining core circadian oscillator function. PPP-1 levels are clock-controlled, peaking in the early evening, and rhythmic PPP-1 levels are necessary for rhythmic P-eIF2α accumulation. Deletion of the N terminus of N. crassa eIF2γ, the region necessary for eIF2γ interaction with GLC7 in yeast, led to high and arrhythmic P-eIF2α levels. These data supported that N. crassa eIF2γ functions to recruit PPP-1 to dephosphorylate eIF2α at night. Thus, in addition to the activity of CPC-3 kinase, circadian clock regulation of eIF2α activity requires dephosphorylation by PPP-1 phosphatase at night. These data show how the circadian clock controls the activity a central regulator of translation, critical for cellular metabolism and growth control, through the temporal coordination of phosphorylation and dephosphorylation events.IMPORTANCE Circadian clock control of mRNA translation contributes to the daily cycling of a significant proportion of the cellular protein synthesis, but how this is accomplished is not understood. We discovered that the clock in the model fungus Neurospora crassa regulates rhythms in protein synthesis by controlling the phosphorylation and dephosphorylation of a conserved translation initiation factor eIF2α. During the day, N. crassa eIF2α is phosphorylated and inactivated by CPC-3 kinase. At night, a clock-controlled phosphatase, PPP-1, dephosphorylates and activates eIF2α, leading to increased nighttime protein synthesis. Translation requires significant cellular energy; thus, partitioning translation to the night by the clock provides a mechanism to coordinate energy metabolism with protein synthesis and cellular growth.

Circadian clock control of eIF2α phosphorylation is necessary for rhythmic translation initiation.

The circadian clock in eukaryotes controls transcriptional and posttranscriptional events, including regulation of the levels and phosphorylation state of translation factors. However, the mechanisms underlying clock control of translation initiation, and the impact of this potential regulation on rhythmic protein synthesis, were not known. We show that inhibitory phosphorylation of eIF2α (P-eIF2α), a conserved translation initiation factor, is clock controlled in Neurospora crassa, peaking during the subjective day. Cycling P-eIF2α levels required rhythmic activation of the eIF2α kinase CPC-3 (the homolog of yeast and mammalian GCN2), and rhythmic activation of CPC-3 was abolished under conditions in which the levels of charged tRNAs were altered. Clock-controlled accumulation of P-eIF2α led to reduced translation during the day in vitro and was necessary for the rhythmic synthesis of select proteins in vivo. Finally, loss of rhythmic P-eIF2α levels led to reduced linear growth rates, supporting the idea that partitioning translation to specific times of day provides a growth advantage to the organism. Together, these results reveal a fundamental mechanism by which the clock regulates rhythmic protein production, and provide key insights into how rhythmic translation, cellular energy, stress, and nutrient metabolism are linked through the levels of charged versus uncharged tRNAs.

Transcriptional repression of frequency by the IEC-1-INO80 complex is required for normal Neurospora circadian clock function.

Rhythmic activation and repression of the frequency (frq) gene are essential for normal function of the Neurospora circadian clock. WHITE COLLAR (WC) complex, the positive element of the Neurospora circadian system, is responsible for stimulation of frq transcription. We report that a C2H2 finger domain-containing protein IEC-1 and its associated chromatin remodeling complex INO80 play important roles in normal Neurospora circadian clock function. In iec-1KO strains, circadian rhythms are abolished, and the frq transcript levels are increased compared to that of the wild-type strain. Similar results are observed in mutant strains of the INO80 subunits. Furthermore, ChIP data show that recruitment of the INO80 complex to the frq promoter is IEC-1-dependent. WC-mediated transcription of frq contributes to the rhythmic binding of the INO80 complex at the frq promoter. As demonstrated by ChIP analysis, the INO80 complex is required for the re-establishment of the dense chromatin environment at the frq promoter. In addition, WC-independent frq transcription is present in ino80 mutants. Altogether, our data indicate that the INO80 complex suppresses frq transcription by re-assembling the suppressive mechanisms at the frq promoter after transcription of frq.

IFT57 stabilizes the assembled intraflagellar transport complex and mediates transport of motility-related flagellar cargo.

Intraflagellar transport (IFT) is essential for the assembly and maintenance of flagella and cilia. Recent biochemical studies have shown that IFT complex B (IFT-B) is comprised of two subcomplexes, IFT-B1 and IFT-B2. The IFT-B2 subunit IFT57 lies at the interface between IFT-B1 and IFT-B2. Here, using a Chlamydomonasreinhardtii mutant for IFT57, we tested whether IFT57 is required for IFT-B complex assembly by bridging IFT-B1 and IFT-B2 together. In the ift57-1 mutant, levels of IFT57 and other IFT-B proteins were greatly reduced at the whole-cell level. However, strikingly, in the protease-free flagellar compartment, while the level of IFT57 was reduced, the levels of other IFT particle proteins were not concomitantly reduced but were present at the wild-type level. The IFT movement of the IFT57-deficient IFT particles was also unchanged. Moreover, IFT57 depletion disrupted the flagellar waveform, leading to cell swimming defects. Analysis of the mutant flagellar protein composition showed that certain axonemal proteins were altered. Taken together, these findings suggest that IFT57 does not play an essential structural role in the IFT particle complex but rather functions to prevent it from degradation. Additionally, IFT57 is involved in transporting specific motility-related proteins.

Regulation of Neurospora Catalase-3 by global heterochromatin formation and its proximal heterochromatin region.

Catalase-3 (CAT-3) constitutes the main catalase activity in growing hyphae of Neurospora crassa, and its activity increases during exponential growth or is induced under different stress conditions. Although extensive progress has been made to identify catalase regulators, the regulation mechanism of CAT-3 at the chromatin level still remains unclear. Here, we aim at investigating the molecular regulation mechanisms of cat-3 at the chromatin level. We found that CAT-3 protein levels increased in mutants defective in proper global heterochromatin formation. Bioinformatics analysis identified a 5-kb AT-rich sequence adjacent to the cat-3 promoter as a heterochromatin region because of its enrichment of H3K9me3 and HP1. Expression of CAT-3 was induced by H2O2 treatment in wild-type and such change occurred along with the accumulation of histone H3 acetylation at 5-kb heterochromatin boundaries and cat-3 locus, but without alteration of its H3K9me3 repressive modification. Moreover, disruption of 5-kb heterochromatin region results in elevated cat-3 expression, and higher levels of cat-3 expression were promoted by the combination with global heterochromatin defective mutants. Interestingly, the molecular weight and activity bands of CAT-3 protein are different in heterochromatin defective mutants compared with those in wild-type, suggesting that its N-terminal processing and modification may be altered. Our study indicates that the local chromatin structure creates a heterochromatin repressive environment to repress nearby gene expression.