Pseudouridine (Psi) is one of the most frequent post-transcriptional modification of RNA. Enzymatic Psi modification occurs on rRNA, snRNA, snoRNA, tRNA, and non-coding RNA and has recently been discovered on mRNA. Transcriptome-wide detection of Psi (Psi-seq) has yet to be performed for the widely studied model organism Drosophila melanogaster. Here, we optimized Psi-seq analysis for this species and have identified thousands of Psi modifications throughout the female fly head transcriptome. We find that Psi is widespread on both cellular and mitochondrial rRNAs. In addition, more than a thousand Psi sites were found on mRNAs. When pseudouridylated, mRNAs frequently had many Psi sites. Many mRNA Psi sites are present in genes encoding for ribosomal proteins, and many are found in mitochondrial encoded RNAs, further implicating the importance of pseudouridylation for ribosome and mitochondrial function. The 7SLRNA of the signal recognition particle is the non-coding RNA most enriched for Psi. The 3 mRNAs most enriched for Psi encode highly expressed yolk proteins (Yp1, Yp2, and Yp3). By comparing the pseudouridine profiles in the RluA-2 mutant and the w1118 control genotype, we identified Psi sites that were missing in the mutant RNA as potential RluA-2 targets. Finally, differential gene expression analysis of the mutant transcriptome indicates a major impact of loss of RluA-2 on the ribosome and translational machinery.
