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

Voytsekh, Olga

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.


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