Circadian clocks respond to temperature changes over the calendar year, allowing organisms to adjust their daily biological rhythms to optimize health and fitness. In, seasonal adaptations and temperature compensation are regulated by temperature-sensitive alternative splicing (AS) of() and() genes that encode key transcriptional repressors of clock gene expression. Although() gene encodes the critical activator of clock gene expression, AS of its transcripts and its potential role in temperature regulation of clock function have not been explored. We therefore sought to investigate whetherexhibits AS in response to temperature and the functional changes of the differentially spliced transcripts. We observed thattranscripts indeed undergo temperature-sensitive AS. Specifically, cold temperature leads to the production of an alternativetranscript, hereinafter termed-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 CK1α-dependent phosphorylation site, is among the four amino acids deleted in CLK-cold protein. Using a combination of transgenic fly, tissue culture, andexperiments, we demonstrated that upon phosphorylation at CLK(S13), CLK-DNA interaction is reduced, thus decreasing CLK occupancy at clock gene promoters. This is in agreement with our findings that CLK occupancy at clock genes and transcriptional output are elevated at cold temperature, which can be explained by the higher amounts of CLK-cold isoforms that lack S13 residue. This study provides new insights into the complex collaboration between AS and phospho-regulation in shaping temperature responses of the circadian clock. Drosophila period per timeless tim clock clk clk clk clk clk in vitro
