Isoform switching facilitates period control in the Neurospora crassa circadian clock

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Ozgur E Akman^1,2,3, James C W Locke^1,4, Sanyi Tang^1,2,3, Isabelle Carré⁵, Andrew J Millar^1,6 & David A Rand^1,2,3

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Synopsis

Circadian rhythms are universal, controlling 24-h rhythms of metabolism, physiology and behaviour in organisms ranging from humans to cyanobacteria. The circadian clock controls the expression of many genes, in a proportion between 10% (in the fruit fly, Drosophila melanogaster) and 100% (in cyanobacteria). Circadian rhythms share similar basic properties—they can be entrained by environmental light and temperature signals, for example. Temperature is interesting as an environmental time cue, and the understanding of its effects has been a central theme of circadian clock research since the 1950s. On the one hand, the clock is sensitive to temperature to the extent that it can act as an entraining signal, while on the other it is insensitive in that the rhythmic period is largely invariant over a physiological range of temperatures. This latter phenomenon, known as temperature compensation, is generally considered to be one of the defining properties of the clock. It has been suggested to be a key requirement for stability of the clock's phase relationship to daily environmental cycles under varying temperatures, and recent work has suggested that the small variation observed is a specific, adaptive control of period. Moreover, given that many biological rates roughly double when temperature is raised by 10°C, it is remarkable that such clocks remain rhythmic over a broad temperature range with period Q₁₀ values in the range 0.8–1.2.

The Neurospora crassa circadian network is particularly interesting from the point of view of temperature regulation because of the discovery that temperature alters the balance of translation between short and long isoforms of the protein FREQUENCY (FRQ), yielding a network with two parallel negative feedback loops that have opposite temperature dependency. A number of different mutant strains have been developed where effectively only one of the forms is present and their temperature responses have been quantified. The evolution of a molecular mechanism to provide a relatively complex modulation of the translation rates of two different forms of the same protein is a fascinating example of the extent to which circadian clocks can diverge from the minimal delayed negative feedback loop network sufficient to produce autonomous, entrainable oscillations exemplified by the Goodwin oscillator.

We introduce a new mathematical model for the N. crassa circadian network, which incorporates recent experimental findings, including the temperature-dependent post-transcriptional modification of the FRQ protein (see Figure 1). This model is used to discuss period control and the functional temperature range of the N. crassa clock. The model reproduces all the key experimental data on temperature dependence and rhythmicity, in both wild-type and mutant strains (some simulations are shown in Figure 3). We present a mechanism (referred to as isoform switching) for period control that utilises the presence of the two parallel, temperature-dependent FRQ loops, suggesting a relatively simple means by which period control, and the consequent buffering of entrainment phase against temperature variations, could have evolved. We argue that this regulatory structure and associated tuning may also increase the temperature range where the clock is robustly rhythmic. Our results support theoretical studies proposing that one of the possible benefits of high loop complexity in clock networks is the increased evolutionary flexibility that such architectures confer. Furthermore, although our period control mechanism is presented in the context of the Neurospora system, we believe that the way in which other systems possessing a temperature-dependent switch in one or more key isoforms achieve compensation and phase robustness may be mathematically similar.

Figure 1

A schematic representation of the regulatory network underlying the mathematical model of the N. crassa clock. This includes the two genes frq and wc-1 and both the long and short forms of the FRQ protein. WC1^* represents light-activated WC-1.

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Figure 3

Dependence of period on temperature for the Neurospora model. Circles denote the WT. Left panel: mutant strains obtained through optimisation or suppression of splicing (Diernfellner et al, 2005, 2007). Inverted triangles: strain A; triangles: strain A with divergent FRQ pathways; diamonds: strain B; squares: strain B with divergent FRQ pathways. For the simulations obtained assuming FRQ pathway asymmetry, strain A has an increasing period–temperature profile, while strain B has a decreasing one with the period of strain A greater than that of strain B, as observed experimentally (Diernfellner et al, 2007). Right panel: mutant strains obtained through modification of the l-FRQ AUG or s-FRQ coding region (Liu et al, 1997). Triangles: strain C; squares: strain D. Strain is compensated at lower temperatures with a period greater than that of the wild-type, becoming arrhythmic at the upper end of the range. Strain D is compensated at higher temperatures with a period smaller than that of the WT, becoming arrhythmic at the lower end of the range. This is in agreement with experimental data (Liu et al, 1997).

Full figure and legend (21K)Figures & Tables index

Finally, by determining the key parameters contributing to period control in the Neurospora clock, we are able to make predictions regarding the biochemical processes affected in compensation mutants associated with changes in FRQ stability, as well as the biological parameters most likely to produce significant changes to the period profile when perturbed experimentally. In particular, our simulations suggest that the FRQ isoforms may have different efficacies as transcription factors, or be differentially phosphorylated, and that changes in the phosphorylation rates of the isoforms will have opposite effects on the period Q₁₀.

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