Fig. 5: Orb2 increases abundance of endogenous rare-codon-enriched mRNAs with annotated Orb2 binding sites in the Drosophila brain.
From: Orb2 enables rare-codon-enriched mRNA expression during Drosophila neuron differentiation
A Scatterplot of annotated Orb2 binding sites and CAI measurements for codon-altered GFP transgenes16 (see Methods). Linear regression reports p value < 0.0001, n = 28. Labeled are GFP-common and GFP-rareb (in bright green). Points are colored by level of expression in Drosophila larvae16: black is ubiquitous, green is tissue-specific, white is none. B Reading frame of Orb2 binding site determines CAI. Shifting the reading frame of Orb2 binding site55,60 can lead to enrichment of rare (green) or common codons (gray). C Overexpressing orb2 isoforms does not enable GFP-rareb expression in NBs. Control (w RNAi), UAS-orb2A and/or UAS-orb2B driven by insc-GAL4. Non-significant between control and all genotypes by One-way ANOVA. Mean ± SEM plotted, precise n values provided in source data file, 3 biological replicates. D Volcano plot of differential RNA expression profiles between insc-GAL4>w RNAi and insc-GAL4>orb2 RNAi. Fold change was plotted as log2(fold change) against adjusted p value, plotted as −log10(adjusted p value). Genes with significantly different fold change are plotted in green for negative and gray for positive. Statistics information provided in methods. E, F Scatter plots of annotated Orb2 binding sites of trnascripts with significantly changed fold change from RNA seq. See methods for details. Linear regression plotted, r and p value for each group shown in the table. E Transcripts upregulated by Orb2. F Transcripts downregulated by Orb2. G Candidate selection criteria for Orb2-regulated transcripts. H, I’ shakB and mGluR mRNA smiFISH in WL3 brains. Dashed lines = NBs, solid lines = neurons. Scale bar 10 µm. One representative image of three animals from one of three biological replicates shown. H-H’ shakB mRNA in insc-GAL4> w-RNAi (H) or insc-GAL4> orb2-RNAi (H’). I–I’. mGluR mRNA in insc-GAL4> w-RNAi (I) or insc-GAL4>orb2-RNAi (I’) J. Plot of change in mRNA abundance over one hour of ActD treatment. Mean ± SEM are plotted. 20 brains each condition, 2 replicates for ND-18 and 3 replicates for mGluR. K, L Plot of mean ± SEM ratio of mRNA over timecourse of ActD treatment with or without orb2. K mGluR mRNA. Two-tailed t test, p = 0.043. L ND-18 mRNA. Two-tailed t test, p value not significant (ns).
