Post-transcriptional regulation of the circadian clock in Ostreococcus tauri

The cellular landscape changes dramatically over the course of a 24 h day. The proteome responds directly to daily environmental cycles and is additionally regulated by the circadian clock. Historically, models of the circadian clock have been built from transcriptomic studies, but over the past few years it has become clear that protein abundance information cannot be assumed from transcriptomic data. To quantify the relative contribution of diurnal versus circadian regulation, we mapped proteome dynamics under light:dark cycles compared with constant light. Using Ostreococcus tauri, a prototypical eukaryotic cell, we achieved 85% coverage, which allowed an unprecedented insight into the identity of proteins that facilitate rhythmic cellular functions. The overlap between diurnally- and circadian-regulated proteins was modest and these proteins exhibited different phases of oscillation between the two conditions. Transcript oscillations were generally poorly predictive of protein oscillations, in which a far lower relative amplitude was observed. We observed coordination between the rhythmic regulation of organelle-encoded proteins with the nuclear-encoded proteins that are targeted to organelles. Rhythmic transmembrane proteins showed a different phase distribution compared with rhythmic soluble proteins, indicating the existence of a circadian regulatory process specific to the biogenesis and/or degradation of membrane proteins. Our observations argue that the cellular spatiotemporal proteome is shaped by a complex interaction between intrinsic and extrinsic regulatory factors through rhythmic regulation at the transcriptional as well as post-transcriptional, translational, and post-translational levels. The most prominent extrinsic factor that might affect the proteome is our planet's 24h light/dark cycle and its effect on the generation of reactive oxygen species (ROS). Redox homeostasis and the circadian clock regulate one another to negate the potential effects of rhythmic light and attain homeostasis. Selenoproteins are an important class of redox-related enzymes that have a selenocysteine residue in the active site. We developed a functional understanding of how environmental and endogenous circadian rhythms integrate to shape the selenoproteome in a model eukaryotic cell. We mined quantitative proteomic data for the 24 selenoproteins of Ostreococcus across time series, again under environmentally rhythmic entrained conditions of light/dark (LD) cycles, compared to constant circadian conditions of constant light (LL). We found an overrepresentation of selenoproteins among rhythmic proteins under LL, but an underrepresentation under LD conditions. Rhythmic selenoproteins under LL that reach peak abundance later in the day showed a greater relative amplitude of oscillations than those that peak early in the day. Under LD, amplitude did not correlate with peak phase; however, we identified high-amplitude selenium uptake rhythms under LD but not LL conditions. Selenium deprivation induced strong qualitative defects in clock gene expression under LD but not LL conditions. Overall, the clear conclusion is that the circadian and environmental cycles exert differential effects on the selenoproteome, and that the combination of the two enables homeostasis. Selenoproteins may therefore play an important role in the cellular response to reactive oxygen species that form as a consequence of the transitions between light and dark. In both the overall proteome and the selenoproteome, the overlap between diurnally-and circadian-regulated proteins was small, and transcript rhythmicity was poorly predictive of protein abundance rhythms. We therefore sought to obtain a circadian RNA-binding proteome to investigate temporal post-transcriptional regulation. We developed a protocol to use UV at 254 nm to covalently crosslink proteins in direct contact with RNA, extract them and then identify the proteins. Alongside the anticipated ribosomal and known RNA-binding proteins, we identified key metabolic enzymes that rhythmically bind RNA under constant circadian conditions. Post-transcriptional regulation by (or of) moonlighting metabolic enzymes must therefore be considered as a potential link reciprocally connecting metabolism to the circadian clock. To ultimately reveal the integration of endogenous and environmental rhythms on the rhythmic flow of information from DNA to rhythmic function, genetic tools are indispensable. While the model cells of Ostreococcus have been instrumental in obtaining detailed knowledge of rhythmicity of the cellular landscape at the -omics level, genetic tools to take the next steps are underdeveloped for this organism. We have made important headway in the set-up of the CRISPR/Cas9 genome editing system in Ostreococcus, to make precise mutations in target gene sequences. We designed two methods for the positive selection of edited cells, and optimised the parameters for the reliable transformation of Cas9 ribonucleoprotein complexes into the algal cells. We designed a diagnostic PCR method for the identification of edited algal DNA, which showed that the genome editing of Ostreococcus can certainly be successful. This thesis provides a step towards an integrated -omics overview of circadian regulation of the eukaryotic cell. Additional temporally resolved datasets, such as for the phosphoproteome and metabolome, taking advantage of the minimal cellular complexity of Ostreococcus, should be collected to fully comprehend the complex interactions between the levels of physiology that together shape cellular rhythmicity.