Alternative splicing (AS) is a dynamic and highly regulated process to expand the proteome diversity by allowing the generation of multiple possible messenger RNA (mRNA) isoforms from a single primary transcript. The splicing process impacts multiple biological processes, including gene expression (GE). By AS the cells can rapidly adapt to changing internal or external stimuli, such as temperature changes. In homeothermic organisms, the core body temperature oscillates in a circadian manner in a range of around 1 °C - 4 °C and these subtle temperature changes are sufficient to control AS. However, whether and how cells can sense and react to these minor changes is not fully understood. In this thesis, we identified the Cdc2-like kinases (CLKs) 1 and 4 as the temperature sensors, which react to changes in the body temperature within a physiologically relevant range with higher activity at lower temperatures. We showed that the mRNA encoding the cold-inducible RNA binding protein (CIRBP) is CLK1/4-dependent alternatively spliced at different temperatures, resulting in a warm-induced isoform with lower stability. Reversible phosphorylation-dependent mechanisms rely on the activity of kinases and phosphatases. In contrast to CLK1/4, we showed the anti-oncogenic protein phosphatase 2A (PP2A) to be more active at higher temperatures. RNA sequencing (RNA-Seq) analyses revealed a global impact of PP2A on AS and GE as phosphatase inhibition using okadaic acid (OA) almost completely abolished temperature sensitivity in HEK293 cells. Besides, the tumor-suppressive transcription factor p53 gets activated at higher temperatures in a PP2A-dependent manner, likely through AS of MDM4. In contrast, oncogenic MYC is more active at lower temperatures, which is consistent with negative regulation of phosphatase activity. These data point to a novel, body temperature-dependent mechanism which activates p53 tumor-suppressive function and provides a molecular mechanism for the use of PP2A inhibitors or hyperthermia in cancer therapies. AS can result in the production of a premature translation termination codon (PTC) and, thereby, control GE by nonsense-mediated mRNA decay (NMD). We found for multiple RNA binding proteins (RBPs) that temperature-dependent alternatively spliced isoforms are often targeted by the NMD pathway leading to body temperature-responsive, rhythmic GE levels. As an example, we showed that the temperature-dependent and NMD-inducing inclusion of SRSF10 exon 3 results in reduced GE levels in a rhythmic manner and suggests that the production of the NMD-targeted isoform is under control of an autoregulatory feedback loop. In SRSF10, splicing of the NMD-inducing, exon 3-containing isoform is under direct competition of a minor and a major splice site in SRSF10 exon 2. Finally, we revealed that SRSF10 autoregulates its expression by activating the inclusion of exon 3. Interestingly, usage of the minor splice site in exon 2, which leads to the production of a coding mRNA, is reduced through the presence of a downstream major splice site leading to AS of the NMD-inducing isoform. We found that SRSF10 transcript levels correlate with the minor spliceosome component RNA binding region (RNP1, RRM) containing 3 (RNPC3) in a tissue- and developmental stage-specific manner. Our data suggest that SRSF10 expression levels control the expression of all other serine/arginine-rich (SR) proteins via cross-regulation. Therefore, the minor spliceosome also controls major intron splicing, which globally affects AS and GE. In summary, this work expands our knowledge of how cells sense and adapt to changes in the body temperature. We showed that the activity of CLK1/4 and PP2A is differently affected by fluctuations in the body temperature, resulting in distinctive AS patterns and expression changes of target genes. We revealed that the temperature-dependent AS-NMD pathway leads to cycling GE and identified the minor spliceosome as a regulator of SRSF10 GE levels, which impacts the expression levels of all SR proteins, thereby globally affecting major intron splicing.
