The Drosophila TART transposon manipulates the piRNA pathway as a counter-defense strategy to limit host silencing

Co-evolution between transposable elements (TEs) and their hosts can be antagonistic, where TEs evolve to avoid silencing and the host responds by reestablishing TE suppression, or mutualistic, where TEs are co-opted to benefit their host. The TART-A TE functions as an important component of Drosophila telomeres, but has also reportedly inserted into the D. melanogaster nuclear export factor gene nxf2. We find that, rather than inserting into nxf2, TART-A has actually captured a portion of nxf2 sequence. We show that Nxf2 is involved in suppressing TART-A activity via the piRNA pathway and that TART-A produces abundant piRNAs, some of which are antisense to the nxf2 transcript. We propose that capturing nxf2 sequence allowed TART-A to target the nxf2 gene for piRNA-mediated repression and that these two elements are engaged in antagonistic co-evolution despite the fact that TART-A is serving a critical role for its host genome.


42
In this study we have characterized the presence of sequence within the coding region of the D. 43 melanogaster nxf2 gene that was previously annotated as an insertion of the TART-A transposon 44 (Sackton et al., 2009). We find that the shared homology between TART-A and nxf2 is actually the 45 result of TART-A acquiring a portion of the nxf2 gene, rather than the nxf2 gene gaining a TART-A 46 insertion. We also find that nxf2 plays a role in suppressing TART-A activity, likely via the piRNA 47 pathway. Our findings support a model where TART-A produces antisense piRNAs that target nxf2 for 48 suppression as a counter-defense strategy in response to host silencing. We identified nxf2 cleavage 49 products from degradome-seq data that are consistent with Aub-directed cleavage of nxf2 transcripts 50 and we find that, across the Drosophila Genetic Reference Panel (DGRP), TART-A copy number is 51 negatively correlated with nxf2 expression. Our findings suggest that TEs can selfishly manipulate host 52 silencing pathways in order to increase their own copy number and that a single TE family can benefit, 53 as well as antagonize, its host genome.

3
The TART-like region of nxf2 is conserved across the melanogaster group 4 It was previously reported that the homology between nxf2 and TART-A is due to an insertion of the 5 TART-A transposable element in the nxf2 gene that became fixed in the ancestor of D. melanogaster 6 and D. simulans (Sackton et al., 2009). To investigate the homology between these elements in more 7 detail, we first extracted 700 bp of sequence from the 3' region of the nxf2 gene that was annotated as 8 a TART-A insertion ( Figure 1A) and used BLAST (Altschul, Gish, Miller, Myers, & Lipman, 1990) to 9 search this sequence against the TART-A RepBase sequence, which was derived from a full-length       contain the nxf2-like sequence. The nxf2-like sequence from these five elements is 100% identical to 41 that from the canonical TART-A sequence. We also identified an additional four TART-A fragments that 42 overlapped with the nxf2-like region. One of the four is also 100% identical to the canonical sequence 43 while the remaining three are between 96%-99% identical to the canonical sequence.

44
We added these nine sequences to the multiple sequence alignment in Figure 1C

4
The nxf2 gene plays a role in suppressing the activity of D. melanogaster telomeric elements 5 Nxf2 is part of an evolutionarily conserved gene family with functions related to export of RNA from the 6 nucleus (Herold et al., 2000). In Drosophila, a paralog of nxf2 (nxf1) has been shown to be involved in 7 the nuclear export of piRNA precursors and the nxf2 gene itself was identified as a member of the

41
We next focused on piRNA production from nxf2. We reasoned that, if nxf2 expression is subject to 42 piRNA-mediated regulation, we should see piRNAs derived from the nxf2 transcript, outside of the 43 region that shares homology with TART-A. We masked the nxf2/TART-A region of shared homology 44 and aligned the piRNA sequence data to the nxf2 transcript. We found low but consistent production of

31
Natural variation in TART-A copy number is correlated with nxf2 expression levels

32
Previous work has shown that there is large variation in HTT element copy number at the telomeres of

44
If the coding sequence of a gene shares sequence homology with a known transposable element, the 45 most likely explanation for this shared homology is that a portion of the gene was derived from a TE

51
Given that viruses and other pathogens have evolved a variety of methods to block or disrupt host 52 defense mechanisms, it is surprising that there is much less evidence for TEs adopting similar 53 strategies (Cosby et al., 2019). However, unlike viruses, TEs depend heavily on vertical transmission 54 from parent to offspring. Any counter-defense strategy that impacts host fitness would therefore 1 decrease the fitness of the TE as well. Furthermore, disruption of host silencing is likely to lead to 2 upregulation of other TEs, making it more likely that will be a severe decrease in host fitness, similar to 3 what is observed in hybrid dysgenesis. These explanations are relevant to our results: TART-A may be 4 targeting nxf2 for its own advantage, but our knockdown experiment shows that nxf2 suppression 5 causes upregulation of many other TEs besides TART-A (Figures 3 and S3) and other studies have appear to be targeting nxf2 in spite of these potentially deleterious consequences? One possibility is 8 that the suppression of nxf2 expression caused by TART-A is relatively mild (i.e. much less than the 9 level of down-regulation caused by the RNAi knockdown), which is enough to provide a slight benefit to 10 TART-A without causing widespread TE activation. It is also possible that the suppression effect was 11 initially much larger, but has since been counterbalanced by cis-acting variants that increase nxf2 14 In summary, our results show that so-called domesticated TEs, if active, can still be in conflict with their 15 host and raise the possibility that TE counter-defense strategies may be more common than previously 16 recognized, despite the potentially deleterious consequences for the host.

TART-A sequence analysis:
We used the TART-A sequence from RepBase (Jurka, 2000), which is           RNAi of the white gene (Bloomington #33613). Seven males of each of these strains were crossed to 1 seven, 3-5 day old, virgin females carrying the nos-GAL4 driver (Bloomington #25751). After 6 days of 2 mating, we discarded the parental flies and then transferred F1 offspring to fresh food for 2.5 days 3 before collecting ovaries from six females for each cross. We performed two biological replicates for 4 each of the three crosses, dissected the ovaries in 1x PBS and immediately transferred them to 5 RNAlater. We extracted RNA using Trizol/Phenol-Chloroform and used the AATI Fragment Analyzer to 6 assess RNA integrity. We then prepared stranded, total RNA-seq libraries by first depleting rRNA with 7 ribo-zero and then using the NEBnext ULTRA II library prep kit to prepare the sequencing libraries. The        degradome-seq data to the same reference sequence used in the piRNA analysis except we unmasked 45 the nxf2 transcript. We analyzed the small RNA data as described under "piRNA analysis" and then 46 used bedtools to extract degradome read alignments whose 5' end was located in the TART-like region 47 of nxf2 and antisense small RNA alignments whose 5' end was located in the nxf2-like region of TART-