Regulation of the Neurospora circadian clock by an RNA helicase - PubMed (original) (raw)
. 2005 Jan 15;19(2):234-41.
doi: 10.1101/gad.1266805. Epub 2004 Dec 29.
Affiliations
- PMID: 15625191
- PMCID: PMC545885
- DOI: 10.1101/gad.1266805
Regulation of the Neurospora circadian clock by an RNA helicase
Ping Cheng et al. Genes Dev. 2005.
Abstract
The eukaryotic circadian oscillators consist of autoregulatory negative-feedback loops. FRQ, WC-1, and WC-2 are three known components of the negative-feedback loop of the Neurospora circadian oscillator. FRQ represses its own transcription by interacting with the WC-1/WC-2 complex and inhibiting WC's role in transcriptional activation. Here we show that all FRQ associates with FRH, an essential DEAD box-containing RNA helicase in Neurospora. The budding yeast homolog of FRH, Dob1p/Mtr4p, is a cofactor of exosome, an important regulator of RNA metabolism in eukaryotes. Down-regulation of FRH by inducible expression of a hairpin RNA leads to low levels of FRQ but high levels of frq RNA and the abolishment of circadian rhythmicities. FRH is associated with the WC complex and this interaction is maintained in a frq null strain. Disruption of the FRQ-FRH complex by deleting a domain in FRQ eliminates the FRQ-WC interaction, suggesting that FRH mediates the interaction between FRQ and the WC complex. These data demonstrate that FRH is an essential component in the circadian negative-feedback loop and reveal an unexpected role of an RNA helicase in regulating gene transcription.
Figures
Figure 1.
Identification of FRH as a FRQ-interacting protein. (A) Silver-stained SDS-PAGE gel showing the affinity purified FRQ–FRH complex. Cell extracts (prepared from LL cultures) of the wild-type and frq10, Myc–FRQ strains were used for purification (described in the Materials and Methods). The Myc–FRQ-containing fractions from a Q-Sepharose column purification were pooled together and immunoprecipitated with the c-Myc monoclonal antibody-coupled agarose beads. (B) The domain structure of the FRH protein.
Figure 2.
All FRQ proteins are in complex with FRH. (A) Immunoprecipitation (IP) assay using the FRH antiserum or the preimmune serum (PI). Extracts of a wild-type strain grown in LL were used. (B) Immunodepletion assay showing that all FRQ proteins are in complex with FRH. IP indicates the antiserum indicated above was used in the assay; PI indicates the preimmune serum was used. (WCL) Whole-cell lysate. (C) Nuclear and cytoplasmic distribution of FRH, FRQ, and WC proteins. (N) Nuclear fraction; (C) cytoplasmic fraction. α-tublin was used as a cytoplasmic control. Equal amounts of nuclear or cytoplasmic proteins were loaded.
Figure 3.
Down-regulation of FRH results in low levels of FRQ protein and an increase of frq RNA level. (A) A schematic diagram showing the inducible gene-silencing approach by expressing a hairpin RNA. (B) Western blot analyses showing the expression of FRQ, FRH, and WC proteins. Cultures were harvested in LL or at DD24 (24 h in DD). The dsfrh strain was used. When QA was used, 1 × 10–2 M of QA was added to the liquid medium. (C) Northern blot analyses showing the expression of frq mRNA for cultures harvested at DD24.
Figure 4.
Down-regulation of FRH results in the loss of circadian conidiation rhythm and FRQ protein rhythms in constant darkness. (A) Race tube assays showing the conidiation rhythms of the dsfrh strain in DD on race tubes containing different concentrations of QA. Black lines indicate the growth fronts every 24 h. (B) Western blot analyses showing the expression of FRQ and FRH in DD in the wild-type strain or the dsfrh strain. Cultures were harvested at the indicated hours in DD. QA was present in the medium for both strains. (C) Densitometric analysis of FRQ Western blot result shown in B.
Figure 5.
(A) Northern blot analyses showing the expression of frq and ccg-2 in DD. Cultures were harvested at the indicated hours in DD. QA was present in the medium for both strains. The RNA samples at DD24 for ccg-2 were mishandled. (B) Northern blot analyses showing the expression of frq in the frq9,dsfrh strain in the presence or absence of QA. Densitometric analysis of the Northern blot result is shown at the bottom. The cultures were grown for 2 d with or without QA in LL before they were transferred into DD.
Figure 6.
FRH interact with the WC complex independent of FRQ. Cultures were harvested in LL. (A,C) Immunoprecipitation assay showing that FRH is associated with the WC complex in a wild-type strain (A) and in a frq null (frq10) strain (C). The IP assays were performed using the WC-2 antiserum (IP) or the preimmune serum (PI). (WCL) Whole cell lysate. (B) IP assay showing the FRQ–FRH interaction in wc-1 or wc-2 mutants expressing QA-inducible FRQ. FRH antiserum was used for IP.
Figure 7.
The interaction between FRQ and FRH is necessary for the complex formation between FRQ and the WC proteins. (A) IP assay showing the loss of FRQ–FRH interaction in the sFRQ6 mutant (frq10,sFRQ6). In the sFRQ3 strain (frq10,sFRQ3), FRQ amino acids 328–422 were deleted from the sFRQ ORF and its FRQ level is comparable to that of sFRQ6. (B) IP assay showing that, in the tFRQ strain, the c-Myc-tagged truncated FRQ protein (FRQ amino acids 656–910) can form a complex with FRH. (C) IP assay showing the loss of FRQ–WC interactions in the sFRQ6 mutant. (D) An updated model of the _Neurospora frq–wc_-based circadian negative-feedback loop.
References
- Aronson B., Johnson, K., Loros, J.J., and Dunlap, J.C. 1994a. Negative feedback defining a circadian clock: Autoregulation in the clock gene frequency. Science 263: 1578–1584. - PubMed
- Bell-Pedersen D., Dunlap, J.C., and Loros, J.J. 1992. The Neurospora circadian clock-controlled gene, ccg-2, is allelic to eas and encodes a fungal hydrophobin required for formation of the conidial rodlet layer. Genes & Dev. 6: 2382–2394. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources