Alternative splicing of Clock transcript mediates the response of circadian clocks to temperature changes

Circadian clocks respond to temperature changes over the calendar year, allowing organisms to adjust their daily biological rhythms to optimize health and fitness. In Drosophila, seasonal adaptations are regulated by temperature- sensitive alternative splicing (AS) of period (per) and timeless (tim) genes that encode key transcriptional repressors of clock gene expression. Although Clock (Clk) gene encodes the critical activator of circadian gene expression, AS of its transcripts and its potential role in temperature regulation of clock function have not been explored. Here, we observed that Clk transcripts undergo temperature- sensitive AS. Specifically, cold temperature leads to the production of an alternative Clk transcript, hereinafter termed Clk-cold, which encodes a CLK isoform with an in- frame deletion of four amino acids proximal to the DNA binding domain. Notably, serine 13 (S13), which we found to be a CK1 alpha- dependent phosphorylation site, is deleted in CLK-cold protein. We demonstrated that upon phosphorylation at CLK(S13), CLK-DNA interaction is reduced, thus decreasing transcriptional activity of CLK. This is in agreement with our findings that CLK occupancy at clock genes and transcriptional output are elevated at cold temperature likely due to higher amounts of CLK-cold isoforms that lack S13 residue. Finally, we showed that PER promotes CK1 alpha- dependent phosphorylation of CLK(S13), supporting kinase- scaffolding role of repressor proteins as a conserved feature in the regulation of eukaryotic circadian clocks. This study provides insights into the complex collaboration between AS and phospho- regulation in shaping temperature responses of the circadian clock.