Transposons contribute to splice-isoform diversity in the Drosophila brain

The extraordinary complexity of the brain depends in part on the vast diversity of mRNA isoforms it expresses, often in a cell-type specific manner. In a recent study, we found that intronic transposable elements (TEs) are spliced into neural transcripts and diversify the splice isoform repertoire of neurons and glia (Treiber and Waddell, 2020). A recent paper by Azad et al. revisits these findings using their TIDAL analysis pipeline applied to our published data (Azad et al., 2024). Their analysis did not find any of the splicing reads we reported, and although they used RT-PCR to test seven of the 264 TE-gene pairs we had previously reported, they failed to validate TE-gene splicing in any of them. Here, we conduct a quantitative analysis of TE exonisation and show that intronic TE insertions are frequently recruited as alternative exons, with exon usage ranging from rare events to near-complete inclusion in transcripts. We implement this analysis in an improved version of our TEChim software, and present clear support for TE-gene splicing at the seven loci tested by Azad et al. We also identify methodological issues in the experimental and computational design of the Azad et al. study that likely explain their failure to detect TE-gene chimeras, while demonstrating that TE-gene splicing can be detected by RT-PCR under appropriate experimental conditions. Together, our data demonstrates that TE splice isoforms are not rare artefacts but measurable and biologically relevant features of the Drosophila brain transcriptome that may contribute to the molecular complexity and functional adaptability of the brain.