Fig. 4: Molecular and cellular evidence of Orb2 as a positive regulator of rare-codon-enriched expression.

A-B’ Images of GFP-rare^b protein (green on left) and mRNA (magenta on left, white on right) in WL3 brains, insc-GAL4>w RNAi (A-A’) or insc-GAL4>orb2 RNAi (B-B’). Scale bar 10 μm. C GFP-rare^b protein decreases with orb2 RNAi in total brains and neurons. Two biological replicates, one-way ANOVA followed by Šídák’s multiple comparison test. w RNAi vs orb2 RNAi total p value = 0.0268. w RNAi vs orb2 RNAi neurons p value < 0.0001. D GFP-rare^b RNA decreases with orb2 RNAi in total brains and neurons. Two biological replicates, w RNAi vs orb2 RNAi total and neurons p < 0.0001 by one-way ANOVA followed by Šídák’s multiple comparison test. E Change in mRNA abundance over one hour of ActD treatment. Mean ± SEM, 20 brains in each condition, 3 replicates for GFP-rare^b and 2 replicates for GFP-common. F, G Plot of ratio of mRNA over 1 hr of ActD treatment with or without orb2. F GFP-rare^b. Two-tailed t test, p = 0.0035. G GFP-common. Two-tailed t test, p not significant (ns). H Schematic of Orb2, CG4612, and CG13928 influencing mRNA polyadenylation and translation. Modified from Khan et al. 2015. I–L Images of GFP-rare^b protein in WL3 brains with insc-GAL4> w RNAi (I), CG4612 RNAi (J), CG13928 RNAi (K). L GFP-rare^b protein expression in total brain for w RNAi, CG4612 RNAi, and CG13928 RNAi normalized to average of all w RNAi brains. Two biological replicates. One-way ANOVA followed by Dunnett’s multiple comparisons test, control vs CG4612 RNAi p = 0.0018. M Schematic of testes shown in N and O and measured in P. µm from tip corresponds to X axis of P. Red = hub, orange = germline stem cell, yellow = spermatogonia, green = spermatocytes, and black = spermatids. N, O. Images of testes GFP fluorescence from mGFP100Dv1 with vasa-GAL4> (N) w RNAi or (O) orb2 RNAi. Scale bar 50 µm. P mGFP100DV1 in testes with vasa-GAL4>w RNAi or orb2 RNAi. Line profiles of GFP-rare^b fluorescence from hub to spermatid (300 μm). N = 9, mean ± SEM, three biological replicates. Welch’s two tailed t test comparing linear regressions p < 0.0001. Precise n values for C, D, and L provided in source data file.