Transposons contribute to splice-isoform diversity in the Drosophila brain
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eLife Assessment
This valuable study addresses a timely question regarding the contribution of transposable elements to splice isoform diversity in the Drosophila brain, directly engaging with recent conflicting findings in the field. The work provides convincing evidence that TE-gene chimeric transcripts are detectable and that prior discrepancies largely arise from methodological differences in computational pipelines and experimental design. The combination of reanalysis, methodological clarification, and targeted validation represents a technical contribution that will be of interest to researchers studying transcriptome complexity and transposable elements. However, the strength of evidence would be further enhanced by increased methodological transparency, more rigorous experimental controls, and a more cautious interpretation of functional implications.
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Abstract
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.
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eLife Assessment
This valuable study addresses a timely question regarding the contribution of transposable elements to splice isoform diversity in the Drosophila brain, directly engaging with recent conflicting findings in the field. The work provides convincing evidence that TE-gene chimeric transcripts are detectable and that prior discrepancies largely arise from methodological differences in computational pipelines and experimental design. The combination of reanalysis, methodological clarification, and targeted validation represents a technical contribution that will be of interest to researchers studying transcriptome complexity and transposable elements. However, the strength of evidence would be further enhanced by increased methodological transparency, more rigorous experimental controls, and a more cautious interpretation of …
eLife Assessment
This valuable study addresses a timely question regarding the contribution of transposable elements to splice isoform diversity in the Drosophila brain, directly engaging with recent conflicting findings in the field. The work provides convincing evidence that TE-gene chimeric transcripts are detectable and that prior discrepancies largely arise from methodological differences in computational pipelines and experimental design. The combination of reanalysis, methodological clarification, and targeted validation represents a technical contribution that will be of interest to researchers studying transcriptome complexity and transposable elements. However, the strength of evidence would be further enhanced by increased methodological transparency, more rigorous experimental controls, and a more cautious interpretation of functional implications.
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Reviewer #1 (Public review):
Summary:
Choucri and Treiber have reassessed their previous study on TE-gene chimeric transcripts in neural genes in response to Azad et al (2024). Azad and colleagues argued that, contrary to Choucri and Treiber's findings, chimeric TE-mRNAs are relatively infrequent, and they cautioned that further optimization of bioinformatics pipelines is needed to detect TE insertions from RNAseq accurately. In this short response, Choucri and Treiber clearly demonstrate that differences in the tools used between their study and that of Azad et al. likely account for the contrasting results, along with RT-PCR failure in designing primers that would match the chimeric transcript, and the use of different Drosophila lines. The authors emphasize the need for uniform, standardized criteria in such analysis, which would …
Reviewer #1 (Public review):
Summary:
Choucri and Treiber have reassessed their previous study on TE-gene chimeric transcripts in neural genes in response to Azad et al (2024). Azad and colleagues argued that, contrary to Choucri and Treiber's findings, chimeric TE-mRNAs are relatively infrequent, and they cautioned that further optimization of bioinformatics pipelines is needed to detect TE insertions from RNAseq accurately. In this short response, Choucri and Treiber clearly demonstrate that differences in the tools used between their study and that of Azad et al. likely account for the contrasting results, along with RT-PCR failure in designing primers that would match the chimeric transcript, and the use of different Drosophila lines. The authors emphasize the need for uniform, standardized criteria in such analysis, which would ultimately strengthen and advance the field.
Strengths:
The addition of a ratio to compute the number of splice reads specific to the chimeric transcript and compare to the exon-exon splice reads is really interesting because it opens the door to finally quantify the contribution of chimeric TEs to the overall gene expression, although this is not the scope of the present article. The clear dissection of chimeric transcripts, along with the results from Azad et al, allows us to understand the differences between the two studies confidently. Finally, the discussion on Drosophila lines is indeed essential, given that the lines and even individuals have high TE polymorphism.
Weaknesses:
I think it is necessary to add more detail to this article, for instance, the differences between TEchim and Tidal could be laid out more precisely. Regarding the roo example, one of the caveats of this family, along with others, is the presence of simple repeats. It would be important to show that the simple repeats are not interfering with the read mapping. Regarding the experiments, if we are looking for a standardized protocol, then we should have a detailed material and methods section, with every experiment, replicate, and PCR temperature clearly defined. Finally, and in my opinion, more importantly, the use of RT negative controls on the RT PCRs, along with DNA PCRs to show insertion presence, is mandatory for testing the presence of chimeric genes. Of course, water negative PCR controls are also needed, and unfortunately, absent from Figure 3.
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Reviewer #2 (Public review):
Summary:
This study by Choucri and Treiber aims to directly address a recent critique regarding the role of transposable elements (TEs) in diversifying the neural transcriptome of Drosophila. The authors seek to demonstrate that TEs are not merely genomic "noise" but are frequently and reliably "exonized" into brain-specific mRNA. By introducing an upgraded computational pipeline, TEChim, and conducting precise experimental validations, the authors set out to show that TE-mediated splicing represents a genuine biological phenomenon that expands the molecular repertoire of the nervous system.
Strengths:
The study's primary strength lies in its rigorous technical "forensic" analysis of previous failed replication attempts. The authors convincingly demonstrate that the lack of signal in the opposing study …
Reviewer #2 (Public review):
Summary:
This study by Choucri and Treiber aims to directly address a recent critique regarding the role of transposable elements (TEs) in diversifying the neural transcriptome of Drosophila. The authors seek to demonstrate that TEs are not merely genomic "noise" but are frequently and reliably "exonized" into brain-specific mRNA. By introducing an upgraded computational pipeline, TEChim, and conducting precise experimental validations, the authors set out to show that TE-mediated splicing represents a genuine biological phenomenon that expands the molecular repertoire of the nervous system.
Strengths:
The study's primary strength lies in its rigorous technical "forensic" analysis of previous failed replication attempts. The authors convincingly demonstrate that the lack of signal in the opposing study stemmed from a fundamental methodological mismatch: the software used by the critics (TIDAL) is logically incapable of detecting splice sites located within TE sequences. Importantly, the authors complement this computational clarification with definitive experimental evidence through an effective "experimental rescue." By employing correctly designed primers and matching the genetic backgrounds of the fly strains, thereby accounting for genomic polymorphisms, they successfully validated all seven loci that were previously reported as undetectable. This dual-pronged strategy, addressing both algorithmic bias and experimental design, establishes a more robust technical benchmark for the detection and validation of TE-derived exons in neural tissues.
Weaknesses:
While the technical rebuttal is highly convincing, the scope of the study remains primarily defensive. As a response to a prior critique, the work focuses on establishing the existence and detectability of chimeric TE-derived transcripts rather than exploring their broader functional consequences. As a result, there is limited new insight into how these TE-modified isoforms influence neural circuit function or organismal behavior. In addition, the detection and validation of these events remain technically demanding, requiring deep sequencing and specialized bioinformatic expertise, which may limit broader adoption by laboratories without dedicated computational resources.
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Reviewer #3 (Public review):
Summary:
This manuscript by Choucri and Treiber responds to a recent paper by Azad et al., which responds to a paper by Treiber and Wadell (Genome Research, 2020). The controversy relates to the detection of transcripts with transposable elements (TEs) spliced into them in the Drosophila brain.
Strengths:
The authors now argue convincingly that these transcripts exist using an improved, updated version of their pipeline. They also validate some of their findings using RT-PCR and explain why Azad et al. failed to detect these transcripts due to methodological errors. Overall, I am convinced that these transcripts exist and that the TE-derived transcripts described by Choucri and Treiber are real.
Weaknesses:
The authors should mention that combining PCR-amplified cDNA generation with short-read sequencing is …
Reviewer #3 (Public review):
Summary:
This manuscript by Choucri and Treiber responds to a recent paper by Azad et al., which responds to a paper by Treiber and Wadell (Genome Research, 2020). The controversy relates to the detection of transcripts with transposable elements (TEs) spliced into them in the Drosophila brain.
Strengths:
The authors now argue convincingly that these transcripts exist using an improved, updated version of their pipeline. They also validate some of their findings using RT-PCR and explain why Azad et al. failed to detect these transcripts due to methodological errors. Overall, I am convinced that these transcripts exist and that the TE-derived transcripts described by Choucri and Treiber are real.
Weaknesses:
The authors should mention that combining PCR-amplified cDNA generation with short-read sequencing is suboptimal for detecting TE-fusion transcripts. Recently, direct long-read ONT RNA sequencing, which does not require amplification and spans the entire transcript, has been used to detect similar transcripts in human stem cells and the human brain (PMID: 40848716 & Garza et al, BioRxiv). Had the authors used this technology to validate their findings, there would be no question about these transcripts. If not doing such experiments, then they should at least discuss the possibility and the advantage of the approach.
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