(A) Diagram of the osk mRNA 3’ UTR. Above the black line (the 3’ UTR) are indicated candidate PTB-binding sites (blue), consisting of any tract of mixed pyrimidines at least 4 nt in length. Mutations within pyrimidine tracts (PTs) that are present in the osk 3’PTmut transgene are indicated in red. Subsets of PT mutations, incorporated into genomic osk transgenes used in panel B, are indicated above (numbers correspond to positions in the osk 3’ UTR). Below the black line, the Bru sites and position of the stem-loop structure containing the RNA dimerization motif are indicated. (B) Subsets of PT mutations disrupt PTB binding to short segments of the osk mRNA 3’ UTR. Short segments of the osk 3’ UTR, each encompassing a subset of PT sites noted in panel A, were used as RNA substrates for UV crosslinking assays with MBP-PTB. The extent of each RNA is indicated above (numbers correspond to positions in the osk 3’ UTR). Radiolabeled RNAs were prepared in wild type form (+), or with the PT mutations from that region (i.e. the same mutations as indicated in red in A)(mut). (C) Subsets of PT mutations disrupt PTB binding to the complete osk mRNA 3’ UTR. UV crosslinking assays with unlabeled competitor RNAs were performed as in Figure 3E, again using the complete osk 3’ UTR as a radiolabeled-binding substrate. The competitor RNAs are indicated above, and consist of the complete osk 3’ UTR with the designated PT mutations. The bar graph below shows the level of residual binding to the radiolabeled substrate RNA at the highest level of competitor (30x molar excess). Smaller values indicate stronger binding of the competitor RNA. Assays were performed three times, and the error bars indicate standard deviations. The 375–540 PTmut RNA was a significantly less effective competitor than the other RNAs with subsets of mutations (p<0.05 for all). (D). Posterior GFP fluorescence from osk::GFP C BRE- when expressed in combination with donor osk mRNAs bearing the indicated mutations. Data are presented as in Figure 3. The student’s t test was used to test for significance of differences relative to the osk+ donor mRNA (left), with the p values shown above. Additional statistical tests evaluated effect size (Cohen’s d) and power. Effect size and power, respectively, for the different donors relative to osk+ were: 3’PTmut, 5.01 and 1.0; 3’159–300 PTmut, 1.53 and >0.99; 3’375–540 PTmut, 4.12 and 1.0; 3’746–789 PTmut, 0.58 and 0.56; 3’853–923 PTmut, 0.88 and 0.91. The statistical significance of the reduced rescuing activity of the 3’746–789 PTmut donor is less compelling than for the other mutants, but this does not invalidate the overall conclusion that the PT mutations have an additive effect on loss of rescuing activity. (E) Levels of donor mRNAs. Values are in comparison to a rescuing osk transgene as in Figure 2. Error bars indicate standard deviations. (F) Posterior GFP fluorescence from osk::GFP (left) or osk::GFP C BRE- (right) when expressed in combination with osk+. Flies were either heph+ or heterozygous for heph1545, as indicated below. The student’s t test was used to test for significance of differences between the two samples in each panel, with the p values shown above. Additional statistical tests evaluated effect size (Cohen’s d) and power. Effect size and power, respectively, were 0.169 and 0.134 (left panel) and 1.12 and >0.99 (right panel).