Fig. 1. Identification of a Nos Response Element in the CycB 3' UTR. (A) Drawing to scale of the CycB 3' UTR and the fragments used in the experiments below. The critical regulatory region (fragment E in red) is present in the maternal mRNA isoform but spliced out of the zygotic isoform (dashed line). Numbers identify the 3' UTR nucleotides present in each fragment. Note that fragment F contains 25 nt encoded by genomic DNA downstream of the polyadenylation and cleavage site. (B) Gel mobility shift experiments with RNA bearing the hb NRE or various fragments of the CycB 3' UTR. On the left, RNA was incubated with embryonic extract prepared as described (Murata and Wharton, 1995). On the right, the Pum RBD was incubated at concentrations of 0, 0.14, 0.42 and 1.3 µM in lanes 1-4 of each titration. The figure is a composite of two different gels. (C) Drawing of the region spanning fragments D and E, where each dot represents a potential Pum-binding site (UGU trinucleotide). The functionally defined 50 nt CycB NRE (black box, nts 594-643 of the 3' UTR) contains two such UGU trinculeotides (red dots, see the sequence in E). Repression in the PGCs at stage 4 for various derivatives is indicated to the right. (D) Accumulation of CycB (green) in stage 4 embryos in which the PGCs are marked by accumulation of Vasa (red), detected by immunofluorescence and confocal microscopy. In these and all subsequent images, repression of CycB mRNA causes the pole cells to appear red, whereas de-repression results in colocalization of CycB and Vasa, causing the pole cells to appear yellow-orange. Embryos are either from w¹¹¹⁸ (wt) nos^BN, or pum^ET3/pum^Msc females, or CycB²/+ females that also bear the indicated CycB transgene. (E) Sequence of the 50 nt CycB NRE (nts 594-643 of the 3' UTR). Binding sites for Nos and Pum are inferred from experiments with purified proteins and a collection of mutant RNAs. Supporting data is presented in Fig. S3 of the supplementary material.