Figure 1: Clk is under strong post-transcriptional regulation

(a) Comparison of clock transcript levels. RNA-seq data⁴¹ were used to compare expression levels of core circadian components in fly heads; an average of six timepoints±s.e. in LD conditions (ZT 2, 6, 10, 14, 18, 22) were analysed. (b) The levels of pre-mRNA and mRNA of core clock components were measured by real-time PCR (RT-PCR) from a mixture of six circadian time points and scaled using an equimolar mixture of the PCR products to obtain absolute measurements between the different mRNAs or pre-mRNAs in each sample (mean±s.e.; three biological replicas). As the values in each sample (pre-mRNA and mRNA) are normalized, the pre-mRNA/mRNA ratio is relative, not absolute (see methods for details). (c) RT-PCR measurements of transcriptional (grey) and steady state (black) levels of Clk and tim. Transcriptional/pre mRNA transcripts were detected using primers for intronic sequences of the genes, whereas steady-state/mature mRNAs were detected using primers flanking two exons. Expression was normalized to RP49 and rpS18. Representative experiment of three repeats is shown. (d) RT-PCR results presenting the ratio of mRNA bound to oligo-dT beads versus unbound mRNA. Data pooled from a mixture of six time points (mean±s.e.; three biological replicates). (e) A Drosophila fly wing system was developed to monitor circadian gene mRNA expression levels (normalized to RP49 at ZT5, 17). Clk and tim levels cycle with the expected phase. The measurements were performed in triplicates for each time point using the Canton-S strain. (f) RT-PCR mRNA half-life measurements for Clk and cry mRNAs from fly wings treated with actinomycin D in five different time points (0–4 h after actinomycin D exposure, 12 h light/12 h dark conditions,mean±s.e.; three biological replicates). See also Supplementary Fig. 1.