Joonseok Cha

Non-optimal codon usage affects expression, structure and function of clock protein FRQ

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 genes. Codon usage is also implicated in the control of transcription, splicing and RNA structure. 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.

Setting the pace of the Neurospora circadian clock by multiple independent FRQ phosphorylation events

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.

The DNA/RNA-dependent RNA polymerase QDE-1 generates aberrant RNA and dsRNA for RNAi in a process requiring replication protein A and a DNA helicase.

The Neurospora RNA-dependent RNA polymerase QDE-1 is an RNA polymerase that can use both RNA and DNA as templates, suggesting a new mechanism for small RNA production.

Control of WHITE COLLAR localization by phosphorylation is a critical step in the circadian negative feedback process.

Reversible protein phosphorylation has critical functions in the eukaryotic circadian negative feedback loops. In Neurospora, the FREQUENCY protein closes the circadian negative feedback loop by promoting the phosphorylation of its transcription activator, the WHITE COLLAR complex (WCC) and consequently inhibiting WCC activity. Here we show that protein phosphatase 4 is a novel component of the Neurospora clock by regulating both processes of the circadian negative feedback loop. The disruption of pp4 results in short period rhythms with low amplitude. In addition to its role in regulating FRQ phosphorylation and stability, PP4 also dephosphorylates and activates WCC. In contrast to PP2A, another phosphatase that activates WCC, PP4 has a major function in promoting nuclear entry of WCC. PKA, a WC kinase, inhibits WC nuclear localization. Furthermore, the FRQ‐dependent WC phosphorylation promotes WCC cytosolic localization. Together, these results revealed WCC nucleocytoplasmic shuttling as an important step in the circadian negative feedback process and delineated the FRQ‐dependent WCC inhibition as a two‐step process: the inhibition of WCC DNA‐binding activity followed by sequestration of WCC into the cytoplasm.

Regulation of the Activity and Cellular Localization of the Circadian Clock Protein FRQ

Eukaryotic circadian clocks employ autoregulatory negative feedback loops to control daily rhythms. In the filamentous fungus Neurospora, FRQ, FRH, WC-1, and WC-2 are the core components of the circadian negative feedback loop. To close the transcription-based negative feedback loop, the FRQ-FRH complex inhibits the activity of the WC complex in the nucleus by promoting the casein kinases-mediated WC phosphorylation. Despite its essential role in the nucleus, most FRQ is found in the cytoplasm. In this study, we mapped the FRQ regions that are important for its cellular localization. We show that the C-terminal part of FRQ, particularly the FRQ-FRH interaction domain, plays a major role in controlling FRQ localization. Both the mutation of the FRQ-FRH interaction domain and the down-regulation of FRH result in the nuclear enrichment of FRQ, suggesting that FRH regulates FRQ localization via a physical interaction. To study the role of FRQ phosphorylation, we examined the FRQ localization in wild-type as well as an array of FRQ kinase, FRQ phosphatase, and FRQ phosphorylation site mutants. Collectively, our results suggest that FRQ phosphorylation does not play a significant role in regulating its cellular localization. Instead, we find that phosphorylation of FRQ inhibits its transcriptional repressor activity in the circadian negative feedback loop. Such an effect is achieved by inhibiting the ability of FRQ to interact with WCC and casein kinase 1a. Our results indicate that the rhythmic FRQ phosphorylation profile observed is an important part of the negative feedback mechanism that drives robust circadian gene expression.

Suppression of WC-independent frequency transcription by RCO-1 is essential for Neurospora circadian clock

Significance Rhythmic clock gene transcription is essential for the functions of eukaryotic circadian clocks. In the Neurospora circadian oscillator, the WHITE COLLAR (WC) complex is responsible for rhythmic frequency (frq) transcription and was thought to be the only transcriptional activator for frq. Here, we show that frq can be constitutively transcribed in a WC-independent manner when the transcriptional corepressor rco-1 is deleted. In rco-1 mutants, high-level constitutive WC-independent frq transcription results in impaired WC activity and loss of circadian rhythmicity. Our results further indicate that RCO-1 acts together with the histone modifier SET-2 and the chromatin remodeling factor CHD-1 to regulate normal chromatin structure at the frq locus, which permits rhythmic frq transcription. Rhythmic activation and repression of clock gene transcription is essential for the functions of eukaryotic circadian clocks. In the Neurospora circadian oscillator, frequency (frq) transcription requires the WHITE COLLAR (WC) complex. Here, we show that the transcriptional corepressor regulation of conidiation-1 (RCO-1) is essential for clock function by regulating frq transcription. In rco-1 mutants, both overt and molecular rhythms are abolished, frq mRNA levels are constantly high, and WC binding to the frq promoter is dramatically reduced. Surprisingly, frq mRNA levels were constantly high in the rco-1 wc double mutants, indicating that RCO-1 suppresses WC-independent transcription and promotes WC complex binding to the frq promoter. Furthermore, RCO-1 is required for maintaining normal chromatin structure at the frq locus. Deletion of H3K36 methyltransferase su(var)3-9-enhancer-of-zeste-trithorax-2 (SET-2) or the chromatin remodeling factor CHD-1 leads to WC-independent frq transcription and loss of overt rhythms. Together, our results uncover a previously unexpected regulatory mechanism for clock gene transcription.

CATP is a critical component of the Neurospora circadian clock by regulating the nucleosome occupancy rhythm at the frequency locus

Rhythmic frq transcription is essential for the function of the Neurospora circadian clock. Here we show that there is a circadian histone occupancy rhythm at the frq promoter that is regulated by FREQUENCY (FRQ). Using a combination of forward genetics and genome sequencing, we identify Clock ATPase (CATP) as an essential clock component. Our results demonstrate that CATP associates with the frq locus and other WCC target genes and promotes histone removal at these loci to allow circadian gene transcription. These results indicate that the rhythmic control of histone occupancy at clock genes is critical for circadian clock function.

Posttranslational control of the Neurospora circadian clock

The eukaryotic circadian clocks are composed of autoregulatory circadian negative feedback loops that include both positive and negative elements. Investigations of the Neurospora circadian clock system have elucidated many of the basic mechanisms that underlie circadian rhythms, including negative feedback and light and temperature entrainment common to all eukaryotic clocks. The conservation of the posttranslational regulators in divergent circadian systems suggests that the processes mediating the modification and degradation of clock proteins may be the common foundation that allows the evolution of circadian clocks in eukaryotic systems. In this chapter, we summarize recent studies of the Neurospora circadian clock with emphasis on posttranslational regulation in the circadian negative feedback loop.

Mechanism of the Neurospora circadian clock, a FREQUENCY-centric view.

Circadian clocks are self-sustaining timekeepers found in almost all organisms on earth. The filamentous fungus Neurospora crassa is a preeminent model for eukaryotic circadian clocks. Investigations of the Neurospora circadian clock system have led to elucidation of circadian clock regulatory mechanisms that are common to all eukaryotes. In this work, we will focus on the Neurospora circadian oscillator mechanism with an emphasis on the regulation of the core clock component FREQUENCY.

Role for Protein Kinase A in the Neurospora Circadian Clock by Regulating White Collar-Independent frequency Transcription through Phosphorylation of RCM-1

ABSTRACT Rhythmic activation and repression of clock gene expression is essential for the eukaryotic circadian clock functions. In the Neurospora circadian oscillator, the transcription of the frequency (frq) gene is periodically activated by the White Collar (WC) complex and suppressed by the FRQ-FRH complex. We previously showed that there is WC-independent frq transcription and its repression is required for circadian gene expression. How WC-independent frq transcription is regulated is not known. We show here that elevated protein kinase A (PKA) activity results in WC-independent frq transcription and the loss of clock function. We identified RCM-1 as the protein partner of RCO-1 and an essential component of the clock through its role in suppressing WC-independent frq transcription. RCM-1 is a phosphoprotein and is a substrate of PKA in vivo and in vitro. Mutation of the PKA-dependent phosphorylation sites on RCM-1 results in WC-independent transcription of frq and impaired clock function. Furthermore, we showed that RCM-1 is associated with the chromatin at the frq locus, a process that is inhibited by PKA. Together, our results demonstrate that PKA regulates frq transcription by inhibiting RCM-1 activity through RCM-1 phosphorylation.