Figure 7. The Pumilio response and AU-rich elements in the her1 3’UTR are both required for rapid reporter transcript turnover.

(A–C) Transgenic embryos carrying the hsp70l:Venus-her1 3’UTR with disrupted PRE & ARE-SV40 pA reporter (line oz93) with a 2 nt mutation in the PRE sequence and 3 nt mutation in the ARE sequence were raised to mid-segmentation stage and heat shocked for 15 minutes, then collected at the indicated minutes pHS and processed by Venus in situ hybridization (n ≥ 11 embryos per time point). Venus transcript is not detected in the absence of heat shock (n = 10 embryos) (data not shown). (D) qPCR analysis comparing Venus transcript fold change from 30 minutes pHS to 60 and 90 minutes pHS for the reporter lines Tg(hsp70l:Venus-her1 3’UTR-SV40 pA)oz44 and Tg(hsp70l:Venus-her1 3’UTR with disrupted PRE & ARE-SV40 pA)oz93 (n = 10 embryos per time point across three biological replicates). The presence of both mutations extends reporter half-life by ~7-fold compared to the unmutated control. Three independent lines carrying the hsp70l:Venus-her1 3’UTR with disrupted PRE & ARE-SV40 pA reporter were analyzed by in situ hybridization and qPCR and each line exhibited comparable Venus decay dynamics (data not shown and Fig. S5); one representative line is shown (see Methods for details). pHS = post-heat shock; PRE = Pumilio response element; ARE = AU-rich element; t_1/2 = half-life; ± = standard deviation; pA = polyadenylation sequence.