The Function of the RNA-Binding Protein CHLAMY1 in the Circadian Clock and its Temperature Integration Process

The research of this thesis focused on the circadian RNA-binding protein CHLAMY1 from the green alga Chlamydomonas reinhardtii that consists of two subunits, C1 and C3. CHLAMY1 binds specifically to (UG)≥7-repeat sequences situated in the 3'-UTRs of several mRNAs such as nitrite reductase 1 (nii1). The role of the C1 subunit within the circadian system was characterized by silencing its gene by an RNAi. The expression level of C1 was silenced down to 25 - 85%. To show the influence of CHLAMY1 on circadian output, NII activities were measured. In wild-type, NII activity peaks around the beginning of subjective day. In the transgenic strain arrhythmicity was observed. Circadian phototaxis was chosen as a second output rhythm. In this case, arrhythmicity was observed immediately or in the first three days under constant conditions in transgenic lines. In addition to these results, a co-regulation between C1 and C3 subunits was found. These data indicate a central role of the C1 subunit in the circadian system of C. reinhardtii. It was also analyzed if the two subunits play a role in temperature integration. C1 was found to be hyper-phosphorylated at 18°C and hypo-phosphorylated at 28°C. The C3 expression level was found to be up-regulated at 18°C, which was shown to occur at the transcriptional level. The clock-relevant CASEIN KINASE1 (CK1) and Ser-/Thr-PROTEIN PHOSPHATASEs (PPs) were found to mediate the temperature dependent regulation of C1 and C3. The expression of CK1 was itself temperature controlled and increased at 28°C. In the long period clock mutant per1 temperature integration of both C1 and C3 was shown to be altered: a low phosphorylation level of C1 and a high expression level of C3 was observed at different temperatures. Altogether, the data suggest that a temperature controlled functional network of clock-relevant proteins exists in C. reinhardtii including C1, C3, CK1, PPs and PER1

Predicting the Physiological Role of Circadian Metabolic Regulation in the Green Alga Chlamydomonas reinhardtii

Although the number of reconstructed metabolic networks is steadily growing, experimental data integration into these networks is still challenging. Based on elementary flux mode analysis, we combine sequence information with metabolic pathway analysis and include, as a novel aspect, circadian regulation. While minimizing the need of assumptions, we are able to predict changes in the metabolic state and can hypothesise on the physiological role of circadian control in nitrogen metabolism of the green alga Chlamydomonas reinhardtii

Both Subunits of the Circadian RNA-Binding Protein CHLAMY1 Can Integrate Temperature Information1[W]

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 up-regulated 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 up-regulation 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 Heteromeric RNA-Binding Protein Is Involved in Maintaining Acrophase and Period of the Circadian Clock

The RNA-binding protein CHLAMY1 from the green alga Chlamydomonas reinhardtii consists of two subunits. One (named C1) contains three lysine homology motifs and the other (named C3) has three RNA recognition motifs. CHLAMY1 binds specifically to uridine-guanine-repeat sequences and its circadian-binding activity is controlled at the posttranslational level, presumably by time-dependent formation of protein complexes consisting of C1 and C3 or C1 alone. Here we have characterized the role of the two subunits within the circadian system by measurements of a circadian rhythm of phototaxis in strains where C1 or C3 are either up- or down-regulated. Further, we have measured the rhythm of nitrite reductase activity in strains with reduced levels of C1 or C3. In case of changes in the C3 level (both increases and decreases), the acrophase of the phototaxis rhythm and of the nitrite reductase rhythm (C3 decrease) was shifted by several hours from subjective day (maximum in wild-type cells) back towards the night. In contrast, both silencing and overexpression of C1 resulted in disturbed circadian rhythms and arrhythmicity. Interestingly, the expression of C1 is interconnected with that of C3. Our data suggest that CHLAMY1 is involved in the control of the phase angle and period of the circadian clock in C. reinhardtii