(A) Graph of the ranking of enrichment of RNAs in AGO-1 immunoprecipitates isolated from flies entrained at 18°C (blue), 25°C (gray), and 29°C (red). Briefly, we ranked RNAs according to their binding enrichment (lower ranking value corresponds to higher binding to AGO-1). To assess the ranking, we averaged the residuals in a linear model: signal in AGO1-immunoprecipitate/signal in input (n = 2 for each temperature). (B) tim isoform-specific qPCR of AGO1-bound mRNAs at 18°C (blue), 25°C (gray), and 29°C (red). Each bar represents an average of all time points normalized to a negative control (cyc). Blue stars indicate statistically significant levels relative to those at 18°C. Significance was determined by two-way ANOVA analysis using Tukey's multiple comparisons test (**, p<0.01; ***, p<0.001). Four different time points from each temperature were used as biological replica. Bars display the means ± SEM. (C) Luciferase assay of whole flies expressing luciferase fused to different tim 3′ UTRs at 18°C (left) and 25°C (right). Blue and gray stars represent statistically significant difference relative to tim-sc or tim-L, respectively, determined by performing two-way ANOVA analysis using Tukey's multiple comparisons test (*, p<0.05; **, p<0.01). Means ± SEM are plotted (n = 8). (D) Table summarizing the number of putative miRNA-binding sites identified in the different tim isoforms using TargetScan. (E) Volcano plot showing miRNAs that have differential expression at 18°C and 29°C (n = 4 for each temperature). The color code represents the significance for each miRNA differentiating all the combinations, for fold change >2 and adjusted p value <0.05. miRNAs with predicted binding sites present in any of the tim isoform 3′ UTRs were identified using TargetScanFly.6.2. Blue, gray, and red indicate putative targets for tim-cold, common sequence, or tim-M 3′ UTRs, respectively. (F) Relative abundances of miRNAs that are differentially expressed (fold change >2 and adjusted p value < 0.05) at different temperatures and that are predicted to target the 3′ UTR of the different isoforms. For each miRNA, the values were normalized to the minimum and their relative expression (RE) is plotted ± SEM. Blue and red stars represent statistical significance relative to expression at 18° or 29°C, respectively, determined using a negative binomial model employing DeSeq2. See details in the 'Materials and methods' (**, p<0.01; ***, p<0.001).