Frequency (gene)
Frequency clock protein | |||||||||
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Identifiers | |||||||||
Symbol | FRQ | ||||||||
Pfam | PF09421 | ||||||||
InterPro | IPR018554 | ||||||||
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Frequency (frq) is a gene encoding the protein frequency (FRQ) that functions in the Neurospora crassa circadian clock. The FRQ protein plays a key role in the auto regulatory transcription-translation negative feedback-loop (TTFL), which is responsible for circadian rhythms in N. crassa as well as in mammals, Drosophila and cyanobacteria.[1]
Discovery
Malcolm L. Sargent, Winslow R. Briggs and Dow O. Woodward at Stanford University discovered the presence of a circadian clock in Neurospora crassa in 1966.[2] They discovered circadian rhythms in asexual spore mutation in one strain of Neurospora called Timex. This strain contained a mutation in the locus band (bd) which was responsible for the circadian spore formation. [3] Strains including the bd locus are now used for all studies of circadian biology in Neurospora.[3] Jerry F. Feldman and Marian N. Hoyle discovered the frq mutant genes (frq-1, frq-2, and frq-3) in 1973.[4] In 1986, frq was first cloned and was shown to rhythmically cycle, which sparked interest in further research and understanding of the N. crassa circadian clock.[5]
Function
![](http://upload.wikimedia.org/wikipedia/en/thumb/4/45/Simplified_Representation_of_Neurospora_Circadian_Clock.jpg/220px-Simplified_Representation_of_Neurospora_Circadian_Clock.jpg)
Frq’s important circadian function is supported by experiments showing that deletion of the frq gene results in arrhythmicity. Frq forms two transcripts that encode two different FRQ proteins, a long form of 989 amino acids (lFRQ) and a shorter form of 890 amino acids (sFRQ).[7] Increased temperature leads to increased expression of lFRQ, while sFRQ is unaffected. This is due to more efficient splicing of an intron in the translation start site in warmer temperatures.[8] Both lFRQ and sFRQ are required for strong rhythmicity. However, the clock is able to persist at certain temperatures, albeit with a weaker rhythmicity, with just one of the proteins present.[9] Since sFRQ favors a longer period than lFRQ, free running rhythms in wild type Neurospora are somewhat decreased with increased temperature.[8] FRQ protein has also been shown to interact with FRH (FRQ-interacting RNA helicase; an essential DEAD box-containing RNA helicase in Neurospora) to form a FRQ/FRH complex (FFC).[10] Overexpression of FRQ protein has been shown to inhibit frq expression, which eventually led to the discovery of the presence of a transcription translation negative feedback loop system in Neurospora.[11] Outputs of the Neurospora circadian clock include carotenoid synthesis and spore formation.[8]
Regulation
![](http://upload.wikimedia.org/wikipedia/en/thumb/e/ef/Frq%2C_FRQ_and_WC1_peaks.jpg/388px-Frq%2C_FRQ_and_WC1_peaks.jpg)
White collar-1 (WC-1) and white collar-2 (WC-2) are GATA transcription factors in Neurospora. Together, they form a heterodimeric "white collar complex" (WCC) via their PAS domains.[13] When WCC is hypophosphorylated during subjective night, it binds to the frequency (frq) gene promoter and activates frq transcription. The frequency (FRQ) protein accumulates and is progressively phosphorylated by casein kinase 1 (CKI), casein kinase 2 (CKII), and a calcium/calmodulin-dependent kinase (CAMK-1), reaching its peak around mid-subjective day.[14] Kinase inhibitors reduce degradation of FRQ by preventing phosphorylation in order to extend the period of the clock.[14] FRQ forms a homodimer to interact with the white collar complex and repress the transcription of frq.[14] When FRQ is hyperphosphorylated, it is ubiquitinated and degraded during the night [8] by the FWD-1 protein, part of the SCF type E3 ligase.[15] FRQ recruits kinases such as casein kinase 1a (CK-1a) that phosphorylates WCC. Hyperphosphorylated WCC is inactive and binds poorly to the frq promoter. This inhibits frq gene transcription and leads to a lower FRQ protein level, and eventually repression is relieved to allow frq transcription to continue and frq mRNA to build up again. This process occurs with a periodicity of around 22 hours in constant conditions.[6][16] FRQ also functions as part of a positive feedback look, separated spatially and temporally from it's repressive activity. In this positive feedback loop, FRQ allows the white collar complex to accumulate.[8]
Mutation
Genetic analysis of the locus has identified alleles that alter the period length of asexual spore formation (conidiation). Mutations have been identified that cause both long and short periods relative to the wild-type value of 21.5 hours. Mutants attain a shorter or longer period due to the rate at which they are phosphorylated. Mutants that can reduce phosphorylation, due to a mutation in a binding site, will not degrade as rapidly and have a longer period. The reverse is true for mutants with a short period.[17] The periods of various frq mutants at 25oC are as follows:
Mutant | frq-1 | frq-2 | frq-3 | frq-4 | frq-6 | frq-7 | frq-8 |
Period (hr) | 16.5 | 19.3 | 24.0 | 19.3 | 19.2 | 29.0 | 29.0 |
The first three mutations are very closely linked since no genetic recombination among them is detected. Each mutant segregates as a single nuclear gene.[18] Of these mutants, the mutations that led to a shorter period (frq-1, frq-2, frq-3, frq-6) did not affect the temperature compensation response. However, the mutants that resulted in a longer period (frq-3, frq-7, frq-8) did show signs of a lower breakpoint temperature, with the lowest breakpoint temperature corresponding to the longest period (29.0 hr).[19] In addition, one recessive allele, frq-9 results in conditional arrhythmicity and a complete loss of temperature compensation.[20]
FRQ-less Oscillator (FLO)
In the absence of a functional frq/white collar oscillator (FWO), Neurospora crassa has still shown oscillation in nitrate reductase activity, diacyglycerol levels, and expression of genes.[21] In frq null mutant Neurospora crassa, a rhythm of conidiospore development was still observed in constant darkness (DD).[22] The period for frq null mutants varied from 12 to 35 hours but could be stabilized by the addition of farnesol or geraniol. However, this mechanism is not well understood.[21] Although the FRQ-less rhythm lost certain clock characteristics such as temperature compensation, temperature pulses were sufficient to reset the clock.[23] There is evidence to support FRQ-less oscillators in Neurospora crassa. For example, many are "slaves" to the frequency/white collar oscillator since they do not possess all of the characteristics of a circadian clock on their own.[21] However, rhythms in clock-controlled gene-16 (ccg-16) are coupled to the FWO but function autonomously, demonstrating that Neurospora crassa contains at least 2 potential pacemakers.[21] The mechanism and significance for FRQ-less oscillators (FLO) are still under research.
Evolution
Since codon optimization of the frq gene results in impaired circadian feedback loop function, frq displays non-optimal codon usage bias across its open reading frame in contrast to most other genes.[24] Casein kinase 2 is conserved in the circadian oscillators of plants (Arabidopsis) and flies (Drosophila).[14] A similar form of casein kinase 1 is necessary for the degradation of period (PER) proteins in Drosophila and mammals.[14] The Drosophila gene slimb is orthologous to FWD1 in Neurospora, both of which are crucial for clock protein degradation.[14]
References
- ^ Lakin-Thomas, PL (March 2000). "Circadian rhythms: new functions for old clock genes?" (PDF). Trends in Genetics. 16 (3): 135–142.
- ^ Sargent ML, Briggs WR, Woodward DO (October 1966). "Circadian nature of a rhythm expressed by an invertaseless strain of Neurospora crassa". Plant Physiol. 41 (8): 1343–9. doi:10.1104/pp.41.8.1343. PMC 550529. PMID 5978549.
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: CS1 maint: multiple names: authors list (link) - ^ a b Belden, W. J., L. F. Larrondo, A. C. Froehlich, M. Shi, C.-H. Chen, J. J. Loros, and J. C. Dunlap. "The Band Mutation in Neurospora Crassa Is a Dominant Allele of Ras-1 Implicating RAS Signaling in Circadian Output." Genes & Development 21.12 (2007): 1494-505. Web.
- ^ Feldman JF, Hoyle MN (December 1973). "Isolation of circadian clock mutants of Neurospora crassa". Genetics. 75 (4): 605–13. PMC 1213033. PMID 4273217.
- ^ McClung CR, Fox BA, Dunlap JC (June 1989). "The Neurospora clock gene Frequency shares a sequence element with the Drosophila clock gene period". Nature. 339 (6225): 558–62. doi:10.1038/339558a0. PMID 2525233.
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: CS1 maint: multiple names: authors list (link) - ^ a b Tseng YY, Hunt SM, Heintzen C, Crosthwaite SK, Schwartz JM (2012). "Comprehensive modelling of the Neurospora circadian clock and its temperature compensation". PLoS Comput. Biol. 8 (3): e1002437. doi:10.1371/journal.pcbi.1002437. PMC 3320131. PMID 22496627.
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: CS1 maint: multiple names: authors list (link) CS1 maint: unflagged free DOI (link) - ^ Nakashima H, Onai K (December 1996). "The circadian conidiation rhythm in Neurospora crassa". Seminars in Cell & Developmental Biology. 7 (6): 765–774. doi:10.1006/scdb.1996.0094.
- ^ a b c d e Axel Diernfellner, Hildur V. Colot, Orfeas Dintsis, Jennifer J. Loros, Jay C. Dunlap, Michael Brunner, Long and short isoforms of Neurospora clock protein FRQ support temperature-compensated circadian rhythms, FEBS Letters, Volume 581, Issue 30, 22 December 2007, Pages 5759-5764, ISSN 0014-5793, http://dx.doi.org/10.1016/j.febslet.2007.11.043. (http://www.sciencedirect.com/science/article/pii/S0014579307011878)
- ^ Liu Y, Garceau NY, Loros JJ, Dunlap JC (May 1997). "Thermally regulated translational control of FRQ mediates aspects of temperature responses in the neurospora circadian clock". Cell. 89 (3): 477–86. doi:10.1016/S0092-8674(00)80228-7. PMID 9150147.
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: CS1 maint: multiple names: authors list (link) - ^ Cheng P, He Q, He Q, Wang L, Liu Y (January 2005). "Regulation of the Neurospora circadian clock by an RNA helicase". Genes Dev. 19 (2): 234–41. doi:10.1101/gad.1266805. PMC 545885. PMID 15625191.
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: CS1 maint: multiple names: authors list (link) - ^ Young, MW; Kay, SA (September 2001). "Time zones: a comparative genetics of circadian clocks". Nature Reviews Genetics. 2: 702–715. doi:10.1038/35088576.
- ^ Dunlap, J. C. et al. “A Circadian Clock in Neurospora: How Genes and Proteins Cooperate to Produce a Sustained, Entrainable, and Compensated Biological Oscillator with a Period of about a Day.” Cold Spring Harbor symposia on quantitative biology 72 (2007): 57–68. PMC. Web. 7 Apr. 2015.
- ^ Cheng P, Yang Y, Liu Y (June 2001). "Interlocked feedback loops contribute to the robustness of the Neurospora circadian clock". Proc. Natl. Acad. Sci. U.S.A. 98 (13): 7408–13. doi:10.1073/pnas.121170298. PMC 34682. PMID 11416214.
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: CS1 maint: multiple names: authors list (link) - ^ a b c d e f He, Q., Cheng, P., Yang, Y., He, Q., Yu, H. and Liu, Y. (2003), FWD1-mediated degradation of FREQUENCY in Neurospora establishes a conserved mechanism for circadian clock regulation. The EMBO Journal, 22: 4421–4430. doi: 10.1093/emboj/cdg425
- ^ Salichos L, Rokas A. The diversity and evolution of circadian clock proteins in Fungi. Mycologia 2010;102:269-78
- ^ Yang Y, Cheng P, He Q, Wang L, Liu Y (September 2003). "Phosphorylation of FREQUENCY protein by casein kinase II is necessary for the function of the Neurospora circadian clock". Mol. Cell. Biol. 23 (17): 6221–8. doi:10.1128/MCB.23.17.6221-6228.2003. PMC 180927. PMID 12917343.
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: CS1 maint: multiple names: authors list (link) - ^ Liu Y, Loros J, Dunlap JC (January 2000). "Phosphorylation of the Neurospora clock protein FREQUENCY determines its degradation rate and strongly influences the period length of the circadian clock". Proc. Natl. Acad. Sci. U.S.A. 97 (1): 234–9. doi:10.1073/pnas.97.1.234. PMC 26646. PMID 10618401.
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: CS1 maint: multiple names: authors list (link) - ^ Dunlap JC (1996). "Genetics and molecular analysis of circadian rhythms". Annu. Rev. Genet. 30: 579–601. doi:10.1146/annurev.genet.30.1.579. PMID 8982466.
- ^ Gardner, George F.; Feldman, Jerry F. (June 1981). "Temperature Compensation of Circadian Period Length in Clock Mutants of Neurospora crassa". Plant Physiology. 68. American Society of Plant Biologists: 1244–1248. doi:10.1104/pp.68.6.1244. PMID 16662086.
- ^ Lewis MT, Feldman JF (November 1996). "Evolution of the Frequency (frq) clock locus in Ascomycete fungi" (PDF). Mol. Biol. Evol. 13 (9): 1233–41. doi:10.1093/oxfordjournals.molbev.a025689. PMID 8896376.
- ^ a b c d Bell-Pedersen, Deborah, Vincent M. Cassone, David J. Earnest, Susan S. Golden, Paul E. Hardin, Terry L. Thomas, and Mark J. Zoran. "Circadian Rhythms from Multiple Oscillators: Lessons from Diverse Organisms."Nature Reviews Genetics 6.7 (2005): 544-56. Web.
- ^ Merrow, M; Roenneberg, T (2007). "Circadian Entrainment of Neurospora crassa". Cold Spring Harb Symp Quant Biol. 72. Cold Spring Harbor Laboratory Press: 279–285. doi:10.1101/sqb.2007.72.032.
- ^ Granshaw T, Tsukamoto M, Brody S (August 2003). "Circadian rhythms in Neurospora crassa: farnesol or geraniol allow expression of rhythmicity in the otherwise arrhythmic strains frq10, wc-1, and wc-2". J. Biol. Rhythms. 18 (4): 287–96. doi:10.1177/0748730403255934. PMID 12932081.
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: CS1 maint: multiple names: authors list (link) - ^ Zhou M, Guo J, Cha J, Chae M, Chen S, Barral JM, Sachs MS, Liu Y (March 2013). "Non-optimal codon usage affects expression, structure and function of clock protein FRQ". Nature. 495 (7439): 111–5. doi:10.1038/nature11833. PMID 23417067.
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: CS1 maint: multiple names: authors list (link)