Both Subunits of the Circadian RNA-Binding Protein CHLAMY1 Can Integrate Temperature Information  

Abstract

The circadian RNA-binding protein CHLAMY1 from the green alga Chlamydomonas reinhardtii consists of two subunits named C1 and C3. Changes in the C1 level cause arrhythmicity of the phototaxis rhythm, while alterations in the level of C3 lead to acrophase shifts. Thus, CHLAMY1 is involved in maintaining period and phase of the circadian clock. Here, we analyzed the roles of the two subunits in the integration of temperature information, the basis for other key properties of circadian clocks, including entrainment by temperature cycles and temperature compensation. Applied temperatures (18°C and 28°C) were in the physiological range of C. reinhardtii. While C1 is hyperphosphorylated at low temperature, the C3 expression level is upregulated at 18°C. An inhibitor experiment showed that this up-regulation occurs at the transcriptional level. Promoter analysis studies along with single promoter element mutations revealed that individual replacement of two DREB1A-boxes lowered the amplitude of c3 up-regulation at 18°C, while replacement of an E-box abolished it completely. Replacement of the E-box also caused arrhythmicity of circadian-controlled c3 expression. Thus, the E-box has a dual function for temperature-dependent upregulation of c3 as well as for its circadian expression. We also found that the temperature-dependent regulation of C1 and C3 as well as temperature entrainment are altered in the clock mutant per1, indicating that a temperature-controlled network of C1, C3, and PER1 exists.

A Flavin Binding Cryptochrome Photoreceptor Responds to Both Blue and Red Light in Chlamydomonas reinhardtii

“…We observed an ;15-fold increase in the C3 transcript after the cells were exposed to 30 min of either blue or red light, demonstrating that the gene encoding C3 belongs to a group of rapidly induced genes such as VVD in Neurospora; a disruption in VVD also causes phase shifts of circadian rhythms (Heintzen et al, 2001;Lewis et al, 2002). Notably, the level of C3 mRNA is strongly influenced by both light and temperature (Voytsekh et al, 2008;Seitz et al, 2010), suggesting that it links light-and temperature-responsive entrainment pathway(s) with the oscillatory system. Moreover, this protein can influence circadian output as part of the CHLAMY1 complex (Kiaulehn et al, 2007).…”

Modeling temperature entrainment of circadian clocks using the Arrhenius equation and a reconstructed model from Chlamydomonas reinhardtii

“…In C. reinhardtii, the circadian RNA-binding protein CHLAMY1 has been shown to induce circadian expression in reporter constructs by binding to (UG) ≥7 -repeats in their 3 -UTR [5]. The phosphorylation of CHLAMY1 subunit C1 is reduced at higher temperature [10]. Changing the expression levels of either subunit of CHLAMY1 causes arhythmicity and phase shifts, respectively [6].…”

“…Changing the expression levels of either subunit of CHLAMY1 causes arhythmicity and phase shifts, respectively [6]. As c3-mRNA as well as the C3 protein cycle over the course of the day, although with very low amplitude at the protein level [10,17,18], it is likely that C3 is part of a negative feedback loop that causes the cycling of its components. In contrast, c1-mRNA and protein levels are relatively constant.…”

“…C1 phosphorylation and the amount of C3 as well as the activity of c3-promotor-driven luciferase reporter constructs vary in a temperature-dependent manner [10]. Both processes might be co-regulated, as phosphorylation of C1 as well as protein levels of C3 and reporter activity are reduced at higher temperature.…”

“…Furthermore, overexpression or silencing of C1 results in an even more pronounced overexpression or silencing of C3, respectively [6,11]. So far it is not known whether temperature compensation or entrainment of the circadian clock or even both processes are influenced by CHLAMY1, but there are indications from per1 mutant studies that at least temperature entrainment is altered in mutants affecting C1 phosphorylation [10]. Furthermore, a role of phosphorylation in temperature regulation has been shown in experiments on other organisms [12,13].…”