Fig. 6

Fig. 6 Download : Download high-res image (509KB)Download : Download full-size image Fig. 6. The Pumilio response and AU-rich elements in the her1 3′UTR contribute to reporter transcript turnover. (A–F) Transgenic embryos carrying the hsp70l:Venus-her1 3′UTR-SV40 pA reporter (line oz44) or the hsp70l:Venus-her1 3′UTR with disrupted PRE-SV40 pA reporter (line oz71) with a 2 ​nt mutation in the PRE sequence were raised to mid-segmentation stage and heat shocked for 15 ​min, 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 per reporter line) (data not shown). (G) qPCR analysis comparing Venus transcript fold change from 30 ​min pHS to 60 and 90 ​min pHS for the reporter lines Tg(hsp70l:Venus-her1 3′UTR-SV40 pA)oz44, Tg(hsp70l:Venus-her1 3′UTR with disrupted PRE-SV40 pA)oz71, and Tg(hsp70l:Venus-her1 3′UTR with disrupted ARE-SV40 pA)oz75 (n ​= ​10 embryos per time point across three biological replicates). The PRE mutation changes the 5′UGUAAAUA site to 5′UCCAAAUA and the ARE mutation changes the 5′UAUUUAU site to 5′UACCCAU. Both mutations extend reporter half-life; the PRE-mutated reporter half-life is increased 1.7-fold and the ARE-mutated reporter half-life is increased 1.6-fold. Five independent lines carrying the hsp70l:Venus-her1 3′UTR with disrupted PRE-SV40 pA reporter and four independent lines carrying the hsp70l:Venus-her1 3′UTR with disrupted ARE-SV40 pA reporter were analyzed in wild-type embryos by in situ hybridization and exhibited comparable Venus decay across all lines carrying the same reporter (data not shown); one representative line for each is shown (see Methods for details). For each reporter, three independent lines were chosen for qPCR analysis and exhibited comparable Venus decay (Supplemental Fig. 3, Supplemental Fig. 4). pHS ​= ​post-heat shock; PRE = Pumilio response element; ARE ​= ​AU-rich element; t_1/2 ​= ​half-life; ± ​= ​standard deviation; pA ​= ​polyadenylation sequence.