Dataset Information

FWD1-mediated degradation of FREQUENCY in Neurospora establishes a conserved mechanism for circadian clock regulation.

ABSTRACT: Phosphorylation of the Neurospora circadian clock protein FREQUENCY (FRQ) regulates its degradation and the proper function of the clock. The mechanism by which FRQ undergoes degradation has not been established. Here we show that FRQ is likely ubiquitylated in vivo, and its proper degradation requires FWD1, an F-box/WD-40 repeat-containing protein. In the fwd1 disruption strains, FRQ degradation is severely impaired, resulting in the accumulation of hyperphosphorylated FRQ. Furthermore, the circadian rhythms of gene expression and the circadian conidiation rhythms are abolished in these fwd1 mutants. Finally, FRQ and FWD1 interact physically in vivo, suggesting that FWD1 is the substrate-recruiting subunit of an SCF-type ubiquitin ligase responsible for FRQ ubiquitylation and degradation. Together with the recent finding that Slimb (the Drosophila homolog of FWD1) is involved in the degradation of the Period protein in flies, our results indicate that FWD1 regulates the degradation of FRQ in Neurospora and is an evolutionarily conserved component of the eukaryotic circadian clock.

SUBMITTER: He Q

PROVIDER: S-EPMC202367 | biostudies-literature | 2003 Sep

REPOSITORIES: biostudies-literature

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FWD1-mediated degradation of FREQUENCY in Neurospora establishes a conserved mechanism for circadian clock regulation.

He Qun Q Cheng Ping P Yang Yuhong Y He Qiyang Q Yu Hongtao H Liu Yi Y

The EMBO journal 20030901 17

Phosphorylation of the Neurospora circadian clock protein FREQUENCY (FRQ) regulates its degradation and the proper function of the clock. The mechanism by which FRQ undergoes degradation has not been established. Here we show that FRQ is likely ubiquitylated in vivo, and its proper degradation requires FWD1, an F-box/WD-40 repeat-containing protein. In the fwd1 disruption strains, FRQ degradation is severely impaired, resulting in the accumulation of hyperphosphorylated FRQ. Furthermore, the cir ...[more]

PMID: 12941694

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Project description:The goal of the experiment was to perform a large scale study of circadian regulation of gene expression in maize. To identify maize genes with expression regulated by the circadian clock, transcript levels in the aerial tissues of young maize seedlings were determined by transcriptional profiling with the Affymetrix GeneChip Maize Genome Array. Maize inbred B73 seedlings were grown inside Conviron growth chamber. B73 seedlings were grown for 7 days under 12 h light:12 h dark (LD) photocycles, 26° C temperature and 70% humidity. At the 8th day, seedlings were transferred to continuous light (LL) and were allowed to entrain completely for 24 h prior to tissue harvest following which tissue was harvested every 4 hours under LL conditions for a period of 48h. Therefore, for the circadian LL time course 12 time points were collected as follows (also defined as factors in the treatment section): ZT0 - 8:00 am/ subjective dawn/ Day1 ZT4 - 12:00 pm/ subjective mid-day/ Day1 ZT8 - 4:00 pm/ subjective late-day/ Day1 ZT12 - 8:00 pm/ subjective dusk/ Day1 ZT16 - 12:00 am/ subjective mid-night/ Day1 ZT20 - 4:00 am/ subjective pre-dawn/ Day1 ZT24- 8:00 am/ subjective dawn/ Day2 ZT28 - 12:00 pm/ subjective mid-day/ Day2 ZT32 - 4:00 pm/ subjective late-day/ Day2 ZT36 - 8:00 pm/ subjective dusk/ Day2 ZT40 - 12:00 am/ subjective mid-night/ Day2 ZT44 - 4:00 am/ subjective pre-dawn/ Day2 Tissue comprised of aerial portion of the seedlings (corresponding to tissue from the prop roots and up) for RNA isolation. Total RNA was isolated from the entire aerial portion of 7 day-old seedlings (corresponding to tissue from the prop roots and up) by Trizol extraction followed by Qiagen RNeasy columns and treatment with RNase-free DNase I (Qiagen; qiagen.com). RNA was isolated from 3 independent biological replicates was pooled. cRNA was generated from pooled total RNA from 3 biological replicates with the GeneChip One-Cycle Target Labeling kit according to the manufacturer’s recommendations (Affymetrix, affymetrix.com). The University of California, Berkeley Functional Genomics Laboratory hybridized samples to Affymetrix GeneChip Maize Genome Arrays and scanned the washed arrays as suggested by manufacturer. Probe sets called “Not Present” or “Marginal” on one or more microarrays were removed from the downstream analysis, as is common practice with circadian studies. Raw hybridization intensities were normalized across all twelve arrays using RMA express in Perfect Match mode. ****[PLEXdb(http://www.plexdb.org) has submitted this series at GEO on behalf of the original contributor, Frank G. Harmon. The equivalent experiment is ZM28 at PLEXdb.] Continuous Light: ZT0(1-replications); Continuous Light: ZT4(1-replications); Continuous Light: ZT8(1-replications); Continuous Light: ZT12(1-replications); Continuous Light: ZT16(1-replications); Continuous Light: ZT20(1-replications); Continuous Light: ZT24(1-replications); Continuous Light: ZT28(1-replications); Continuous Light: ZT32(1-replications); Continuous Light: ZT36(1-replications); Continuous Light: ZT40(1-replications); Continuous Light: ZT44(1-replications)

Dataset Information

FWD1-mediated degradation of FREQUENCY in Neurospora establishes a conserved mechanism for circadian clock regulation.

Publications

FWD1-mediated degradation of FREQUENCY in Neurospora establishes a conserved mechanism for circadian clock regulation.

Similar Datasets

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