Functional significance of FRH in regulating the phosphorylation and stability of Neurospora circadian clock protein FRQ

Abstract

FREQUENCY (FRQ) is the central component of the Neurospora circadian clock. All FRQ proteins form the FFC complex with FRH (FRQ-interacting RNA helicase) that acts as the negative element in the circadian negative feedback loop by repressing frq mRNA levels. To understand the function of the FRQ-FRH interaction, we mapped and identified the minimal FRQ region that is required for FRQ-FRH interaction. We demonstrated that the FRQ-FRH complex formation is required for the interaction between FRQ and the White Collar Complex (WCC) and clock function. On the other hand, in the FRQ-FRH complex, FRQ is also required for the FRH-WCC interaction. Disruption of FRQ-FRH interaction or down-regulation of FRH results in hypophosphorylation, rapid degradation of FRQ, as well as low levels of WHITE COLLAR-1 and WHITE COLLAR-2. Furthermore, we showed that the rapid FRQ degradation in the absence of FRH is independent of FWD-1, the ubiquitin E3 ligase of FRQ under normal conditions, thus uncovering an alternative pathway for FRQ degradation.

FIGURE 1.

Mapping of the minimal region for FFC association. A, sequence alignments showing the sequence consensus of FRQ proteins from various fungi as well as respective mutated regions or residues of sFRQ6 mutants. Residues labeled with “A” above were mutated to alanine. B–D, immunoprecipitation results using FRH antiserum showing the interaction between FRH and FRQ in different FRQ mutants. PI, preimmune serum used in IP. wt, wild type. N. crassa, Neurospora crassa; Sordaria fimicola, S. fimicola; Lyreidus australiensis, L. australiensis; Chaunus spinulosus, C. spinulosus; Gibberella zeae, G. zeae; Magnaporthe grisea, M. grisea; Pred. sec. struc., predicted secondary structure.

FIGURE 2.

FRH regulates the association between FRQ and WCC. A and B, IP results show that WC-2 fails to interact with FRQ or FRH in the FRQ6B2 and FRQ6B5 mutants, by using WC-2 antiserum or preimmune serum (PI). wt, wild type. B, longer exposure of FRH is also presented. C, race tube assays of FRQ6B mutants. The strains were grown in constant darkness (DD) in race tubes, and the growth fronts were labeled every 24 h. The race tubes shown here are representative samples from six replicate tubes.

FIGURE 3.

FRQ participates in regulation of the FRH-WCC interaction. A, IP results show that WC-2 binds to FRQ, FRH, and WC-1 in the FRQ6B1, FRQ6B3, and FRQ6B4 mutants. WC-2 antibody was used in the IP assays and the preimmune serum for the PI sample. WT, wild type. B, IP results showing FRH binds to FRQ in both frq⁹ Myc-FRQ165RR strain and frq⁹ Myc-FRQ strains. FRH antibody was used for the IP assays and preimmune serum for the PI sample. FRH antiserum and c-Myc antibody were used for the Western blot analyses.

FIGURE 4.

Hypophosphorylation of FRQ in the FRQ6 mutants and dsfrh strain. A, hypophosphorylation of FRQ in FRQ6B2, FRQ6B5, mutants, and dsfrh strain. For comparison, total proteins of the mutants were loaded more than wild type (wt) in the Western blot analyses due to the low FRQ protein levels in the mutants. B, hypophosphorylation of FRQ in wild type (WT) and dsfrh strains by QA treatment. Wild-type strain treated with QA served as control. Asterisk denotes nonspecific bands due to long exposure.

FIGURE 5.

Rapid degradation of FRQ protein in the sFRQ6 mutants and dsfrh strain. A, degradation of FRQ in the FRQ6B2 and FRQ6B5 strains. The frq¹⁰,kaj120 strain was used as the control. B, degradation of FRQ in the dsfrh strain with/without QA treatment. The stability of FRQ was also compared in wild type (wt) strain with/without addition of QA, which indicated that QA is not the cause for the change of FRQ degradation rate. Membranes (mem) stained with Amido Black were used as loading control. Cycloheximide was added to inhibit the protein translation (10 μg/ml final concentration). Data are means ± S.D., n = 3. Asterisks indicate p < 0.05.

FIGURE 6.

Abolishment of FRH-FRQ association resulted in decrease of WC proteins. A–F, Western blot analysis results showing the levels of WCs in the indicated strains. Amido Black-stained membranes were used as loading control. WT, wild type.

FIGURE 7.

FRH regulates FRQ stability independent of FWD-1. A, race tube tests of fwd-1^RIP,dsfrh. The growth was inhibited in the presence of QA (10⁻³ m). B, Western blot analysis showing that FRH was silenced in fwd-1^RIP,dsfrh strain by QA treatment (10⁻² m). Nonspecific bands served as control. C, FRQ protein was hypophosphorylated and dramatically reduced in fwd-1^RIP,dsfrh strain in the presence of QA (10⁻² m). D, FRQ protein exhibited rapid degradation in the fwd-1^RIP,dsfrh strain after QA treatment (10⁻² m). Membranes stained with Amido Black were used as loading control. Cycloheximide was added to inhibit the protein translation (10 μg/ml final concentration). Data are mean ± S.D., n = 3. Asterisks indicate p < 0.05. WT, wild type; mem, membrane.