Figure 6.

B52 stimulates dmpi8 splicing in flies. (a,b) Crosses were set up between the TUG driver and independent lines of RNAi-B52 (V38862, V101740 and T37519). For each RNAi line, we set up two crosses; in one set we used male TUG and female virgin RNAi lines and in the other set we did a reciprocal cross. For each cross, young adult progeny were placed in 6 vials (each vial had ~40 flies), and entrained for 5 days in 12 hr:12 hr light/dark (LD) cycles at 25 °C. In addition, the same was done for the parental controls (TUG, V38862, V101740 and T37519). On the last day of LD, at the indicated times, 1 vial for each cross was collected by freezing. For each RNAi line, flies from both crosses were pooled. Total RNA was extracted from fly heads and the splicing efficiency of dmpi8 (a) and total levels of dper (b) were calculated. Because results were highly similar for the different RNAi-B52 line and their parental controls (Fig. S5), we averaged all three TUG > RNAi-B52 crosses, and pooled the data from the parental controls to yield the group averages shown. Values shown for dmpi8 splicing efficiency (shown as fraction, where 1.0 is equal to 100% splicing of dmpi8) and dper RNA levels are from the average of two independent experiments. Note that the values for ZT0 were re-plotted for ZT24. (a) The daily dmpi8 splicing efficiency for flies expressing RNAi-B52 in tim-expressing cells (TUG > RNAi-B52) was significantly different compared to the parental controls (ANOVA, p = 0.010); in addition, for each time point we determined p values (two-tailed t-test); ZT0/24, 0.027; ZT4, 0.0054; ZT8, 0.45; ZT12, 0.0051; ZT16, 0.031; ZT20, 0.026. (b) For dper RNA levels we determined p values for each time point (two-tailed t-test); ZT0/24, 0.23; ZT4, 0.15; ZT8, 0.009; ZT12, 0.004; ZT16, 0.23; ZT20, 0.60. *p < 0.05; **p < 0.01; two-tailed t-test. The data clearly show that knock down of B52 in tim-expressing cells reduces the daily splicing efficiency of dmpi8.