The frq gene, essential for circadian clock function, is shown to differ from most other genes in Neurospora by exhibiting non-optimal codon usage; by contrast, optimization of codon usage is unexpectedly found to affect the structure and function of the coded protein, subsequently impairing circadian feedback loops. Many biological processes are rhythmically regulated on a daily, or circadian, cycle. Highly expressed genes, such as those regulating the circadian rhythm, normally contain optimal codons, to allow efficient expression. Two studies, from the laboratories of Carl Johnson and Yi Liu, now find that central circadian proteins in cyanobacteria and Neurospora unexpectedly use non-optimal codons, and that optimizing their codes causes a change in an adaptive response in cyanobacteria, and compromises function of the Neurospora clock. These works highlight an unanticipated selection against optimal codon usage. Codon-usage bias has been observed in almost all genomes and is thought to result from selection for efficient and accurate translation of highly expressed genes1,2,3. Codon usage is also implicated in the control of transcription, splicing and RNA structure4,5,6. Many genes exhibit little codon-usage bias, which is thought to reflect a lack of selection for messenger RNA translation. Alternatively, however, non-optimal codon usage may be of biological importance. The rhythmic expression and the proper function of the Neurospora FREQUENCY (FRQ) protein are essential for circadian clock function. Here we show that, unlike most genes in Neurospora, frq exhibits non-optimal codon usage across its entire open reading frame. Optimization of frq codon usage abolishes both overt and molecular circadian rhythms. Codon optimization not only increases FRQ levels but, unexpectedly, also results in conformational changes in FRQ protein, altered FRQ phosphorylation profile and stability, and impaired functions in the circadian feedback loops. These results indicate that non-optimal codon usage of frq is essential for its circadian clock function. Our study provides an example of how non-optimal codon usage functions to regulate protein expression and to achieve optimal protein structure and function.

The eukaryotic circadian oscillators consist of circadian negative feedback loops. In Neurospora, it was proposed that the FREQUENCY (FRQ) protein promotes the phosphorylation of the WHITE COLLAR (WC) complex, thus inhibiting its activity. The kinase(s) involved in this process is not known. In this study, we show that the disruption of the interaction between FRQ and CK-1a (a casein kinase I homolog) results in the hypophosphorylation of FRQ, WC-1, and WC-2. In the ck-1a^L strain, a knock-in mutant that carries a mutation equivalent to that of the Drosophila dbt^L mutation, FRQ, WC-1, and WC-2 are hypophosphorylated. The mutant also exhibits ~32 h circadian rhythms due to the increase of FRQ stability and the significant delay of FRQ progressive phosphorylation. In addition, the levels of WC-1 and WC-2 are low in the ck-1a^L strain, indicating that CK-1a is also important for the circadian positive feedback loops. In spite of its low accumulation in the ck-1a^L strain, the hypophosphorylated WCC efficiently binds to the C-box within the frq promoter, presumably because it cannot be inactivated through FRQ-mediated phosphorylation. Furthermore, WC-1 and WC-2 are also hypophosphorylated in the cka^RIP strain, which carries the disruption of the catalytic subunit of casein kinase II. In the cka^RIP strain, WCC binding to the C-box is constantly high and cannot be inhibited by FRQ despite high FRQ levels, resulting in high levels of frq RNA. Together, these results suggest that CKI and CKII, in addition to being the FRQ kinases, mediate the FRQ-dependent phosphorylation of WCs, which inhibit their activity and close the circadian negative feedback loop.

Summary: Nickel serves as a cofactor for various microbial enzymes including superoxide dismutase (SOD) found in Streptomyces spp. In Streptomyces coelicolor, nickel represses and induces production of Fe‐containing and Ni‐containing SODs, respectively, primarily at the transcriptional level. We identified the nickel‐responsive regulator (Nur), a Fur (ferric‐uptake regulator) homologue, which binds to the promoter region of the sodF gene encoding FeSOD in the presence of nickel. Disruption of the nur gene caused constitutive expression of FeSOD and no induction of NiSOD in the presence of nickel. The intracellular level of nickel was higher in a Δnur mutant than in the wild type, suggesting that Nur also regulates nickel uptake in S. coelicolor. A putative nickel‐transporter gene cluster (nikABCDE) was identified in the genome database. Its transcription was negatively regulated by Nur in the presence of nickel. Purified Nur protein bound to the nikA promoter region in a nickel‐dependent way. These results support the action of Nur as a regulator of nickel homeostasis and antioxidative response in S. coelicolor, and add a novel nickel‐responsive member to the list of versatile metal‐specific regulators of the Fur family.

Protein phosphorylation plays essential roles in eukaryotic circadian clocks. Like PERIOD in animals, the Neurospora core circadian protein FRQ is progressively phosphorylated and becomes extensively phosphorylated before its degradation. In this study, by using purified FRQ protein from Neurospora , we identified 43 in vivo FRQ phosphorylation sites by mass spectrometry analysis. In addition, we show that CK-1a and CKII are responsible for most FRQ phosphorylation events and identify an additional 33 phosphorylation sites by in vitro kinase assays. Whole-cell metabolic isotope labeling and quantitative MS analyses suggest that circadian oscillation of the FRQ phosphorylation profile is primarily due to progressive phosphorylation at the majority of these newly discovered phosphorylation sites. Furthermore, systematic mutations of the identified FRQ phosphorylation sites led to either long or short period phenotypes. These changes in circadian period are attributed to increases or decreases in FRQ stability, respectively. Together, this comprehensive study of FRQ phosphorylation reveals that regulation of FRQ stability by multiple independent phosphorylation events is a major factor that determines the period length of the clock. A model is proposed to explain how FRQ stability is regulated by multiple phosphorylation events.

Regulation of circadian clock components by phosphorylation plays essential roles in clock functions and is conserved from fungi to mammals. In the Neurospora circadian negative feedback loop, FREQUENCY (FRQ) protein inhibits WHITE COLLAR (WC) complex activity by recruiting the casein kinases CKI and CKII to phosphorylate the WC proteins, resulting in the repression of frq transcription. On the other hand, CKI and CKII progressively phosphorylate FRQ to promote FRQ degradation, a process that is a major determinant of circadian period length. Here, by using whole-cell isotope labeling and quantitative mass spectrometry methods, we show that the WC-1 phosphorylation events critical for the negative feedback process occur sequentially—first by a priming kinase, then by the FRQ-recruited casein kinases. We further show that the cyclic AMP-dependent protein kinase A (PKA) is essential for clock function and inhibits WC activity by serving as a priming kinase for the casein kinases. In addition, PKA also regulates FRQ phosphorylation, but unlike CKI and CKII, PKA stabilizes FRQ, similar to the stabilization of human PERIOD2 (hPER2) due to the phosphorylation at the familial advanced sleep phase syndrome (FASPS) site. Thus, PKA is a key clock component that regulates several critical processes in the circadian negative feedback loop.

The production of aberrant RNA (aRNA) is the initial step in several RNAi pathways. How aRNA is produced and specifically recognized by RNA-dependent RNA polymerases (RdRPs) to generate double-stranded RNA (dsRNA) is not clear. We previously showed that in the filamentous fungus Neurospora, the RdRP QDE-1 is required for rDNA-specific aRNA production, suggesting that QDE-1 may be important in aRNA synthesis. Here we show that a recombinant QDE-1 is both an RdRP and a DNA-dependent RNA polymerase (DdRP). Its DdRP activity is much more robust than the RdRP activity and occurs on ssDNA but not dsDNA templates. We further show that Replication Protein A (RPA), a single-stranded DNA-binding complex that interacts with QDE-1, is essential for aRNA production and gene silencing. In vitro reconstitution assays demonstrate that QDE-1 can produce dsRNA from ssDNA, a process that is strongly promoted by RPA. Furthermore, the interaction between QDE-1 and RPA requires the RecQ DNA helicase QDE-3, a homolog of the human Werner/Bloom Syndrome proteins. Together, these results suggest a novel small RNA biogenesis pathway in Neurospora and a new mechanism for the production of aRNA and dsRNA in RNAi pathways. Small RNA molecules (20–30 nucleotides) play important roles in many cellular processes in eukaryotic organisms by silencing gene expression. To generate the many forms of small RNAs, DNA is first transcribed to produce single-stranded RNA (ssRNA), which then is converted to double-stranded RNA (dsRNA) by an RNA-dependent RNA polymerase (RdRP). However, it is not clear how the ssRNA templates are synthesized from DNA and specifically recognized by RdRPs amidst a sea of single-stranded, cellular RNAs. We previously showed that in the filamentous fungus Neurospora the production of one type of small RNA called qiRNA, which is specifically induced after DNA damage, requires the RdRP QDE-1. Here, we investigated the precise contributions of QDE-1 to the synthesis of ssRNA and dsRNA. We show that QDE-1 is surprisingly promiscuous in its template choice in that it is able to synthesize RNA from both ssRNA and single-stranded DNA (ssDNA). These results suggest that QDE-1 first generates ssRNA from a DNA template and then converts the ssRNA into dsRNA; this combination of activities in one protein ensures the specific action by RdRP on aberrant RNA in lieu of other single-stranded cellular RNA. In addition, we identified Replication Protein A, a ssDNA-binding protein that interacts with QDE-1, as an essential factor for small RNA production. Furthermore, we were able to reconstitute synthesis of dsRNA from ssDNA in a test tube using purified QDE-1 and RPA proteins, demonstrating the ability of this relatively simple biosynthetic system to generate the nucleic acid trigger for gene regulation. Together, these results uncover the details of a new and important small RNA production mechanism in cells.