Alternative pre-mRNA splicing allows the generation of multiple mRNA isoforms from one precursor. Translation of these isoforms drastically increases the structural and functional space of the proteome. Besides the expansion of protein diversity, alternative splicing creates an additional layer of gene expression regulation, as it influences several steps of the mRNA maturation process. Alternative splicing is a very fast process and therefore represents an ideal mechanism for cells to quickly respond to changed internal or external conditions. Temperature is an omnipresent external clue, influencing basically every aspect of life. The ability of organisms to sense potentially noxious temperatures is a fundamental requirement to survive. Homeothermic organisms are able to keep their core body temperature constant, which makes them resilient to even strongly fluctuating external temperatures. However, the core body temperature itself also slightly oscillates in a time of the day dependent manner. Within this thesis we showed that these subtle core body temperature oscillations are sufficient and necessary to control time of the day dependent alternative splicing of a large and functionally related group of genes in a concerted manner. We further demonstrated that this splicing program is controlled by temperature-dependent phosphorylation of SR proteins. Overexpression and knockdown experiments suggested a role of Cdc2-like kinases (CLKs), a family of SR protein specific kinases, in the regulation of temperature-dependent SR protein phosphorylation. In in vitro kinase assays we showed that activity of human and mouse CLKs reacts extremely sensitive to temperature changes within a physiological relevant temperature range with higher activity at lower temperatures, therefore matching the in vivo phosphorylation state of SR proteins. The temperature dependence is an inherent feature of the kinase domain and is caused by slight conformational changes within the activation segment, as shown by molecular dynamics simulations and biochemical assays. Furthermore, CLK temperature dependence is specific, as activity of a member of the other family of SR protein kinases, namely SRPK1, was stable in the contemplated temperature range. We further investigated the role of CLKs as in vivo thermometers by using RNA-sequencing and demonstrated that inhibition with the specific CLK inhibitor TG003 results in an almost complete loss of temperature-dependent alternative splicing in HEK293 cells. Additionally, temperature-dependent gene expression was markedly reduced upon CLK inhibition. CLKs are conserved throughout eukaryotic evolution, which is why we focused on the thermo-sensing ability of CLK homologs from different species. All tested homologs exhibited a temperature profile exactly fitting the respective living temperatures with a drop of activity at the upper limit. Furthermore, the dynamic temperature range of reptilian CLK homologs suggests a role in temperature-dependent sex determination. Lastly, we focused on a CLK homolog from Cyanidioschyzon merolae (LlK), a red algae inhabiting hot springs. The temperature activity optimum of the LlK kinase domain also exactly fell into the preferred living temperature of C. merolae with a maximum activity at 48 °C and a subsequent drop of activity at the upper temperature limit. We further sought to crystallize its kinase domain and were able to obtain a structure at 2.5 Å resolution. We identified a salt bridge, which directly contributes to heat-stability and temperature-dependent activity of the kinase, as shown by mutational analysis, in vitro kinase assays and CD spectroscopy. In summary, this thesis offers comprehensive insights of how subtle changes in body or ambient temperatures are sensed and integrated into altered alternative splicing and gene expression programs by CLKs.
