Repeated duplication of Argonaute2 is associated with strong selection and testis specialization inDrosophila

Abstract

Argonaute2 (Ago2) is a rapidly evolving nuclease in the Drosophila melanogaster RNA interference (RNAi) pathway that targets viruses and transposable elements in somatic tissues. Here we reconstruct the history of Ago2 duplications across the D. obscura group and use patterns of gene expression to infer new functional specialization. We show that some duplications are old, shared by the entire species group, and that losses may be common, including previously undetected losses in the lineage leading to D. pseudoobscura. We find that while the original (syntenic) gene copy has generally retained the ancestral ubiquitous expression pattern, most of the novel Ago2 paralogs have independently specialized to testis-specific expression. Using population genetic analyses, we show that most testis-specific paralogs have significantly lower genetic diversity than the genome-wide average. This suggests recent positive selection in three different species, and model-based analyses provide strong evidence of recent hard selective sweeps in or near four of the six D. pseudoobscura Ago2 paralogs. We speculate that the repeated evolution of testis specificity in obscura group Ago2 genes, combined with their dynamic turnover and strong signatures of adaptive evolution, may be associated with highly derived roles in the suppression of transposable elements or meiotic drive. Our study highlights the lability of RNAi pathways, even within well-studied groups such as Drosophila, and suggests that strong selection may act quickly after duplication in RNAi pathways, potentially giving rise to new and unknown RNAi functions in nonmodel species. KEYWORDS Argonaute; RNAi; Drosophila; duplication; testis A RGONAUTE genes are found in almost all eukaryotes, where they play a key role in antiviral immune defense, gene regulation, and genome stability. They perform this diverse range of functions through their role in RNA interference (RNAi) mechanisms, an ancient system of nucleic acid manipulation in which small RNA (sRNA) molecules guide Argonaute proteins to nucleic acid targets through base complementarity (reviewed in Meister 2013). Gene duplication has occurred throughout the evolution of the Argonaute gene family, with ancient duplication events characteristic of some lineages-such as three duplications early in plant evolution (Singh et al. 2015) and multiple expansions and losses throughout the evolution of nematodes (reviewed in Buck and Blaxter 2013) and the Diptera . After duplication, Argonautes have often undergone functional divergence, involving changes in expression patterns and altered sRNA binding partners (Lu et al. 2011;Leebonoi et al. 2015;Miesen et al. 2015). Duplication early in eukaryotic evolution produced two distinct Argonaute subfamilies, Ago and Piwi, which have since been retained in the vast majority of Metazoa (Cerutti and Casas-Mollano 2006). Members of the Ago subfamily are expressed in both somatic and germline tissue, and variously bind sRNAs derived from host transcripts (miRNAs, endo-siRNAs) ...

Widespread gene duplication and adaptive evolution in the RNA interference pathways of the Drosophila obscura group

“…but one of the resulting duplicates becoming testis-specific, and most displaying evidence of recent and/or ongoing positive selection [4].…”

“…pseudoobscura [4]. This is particularly striking in the obscura group of Drosophila species [27], which has experienced at least 6 independent duplications of Ago2 over the last 20 million years, with all…”

“…Under sub-functionalization, the duplicates independently accumulate mutations that allow them to specialise in a subset of ancestral functions of a pleiotropic gene. Neo-functionalization leads to asymmetrical evolutionary rates among paralogs (with faster evolution in paralogs that gain derived function), whereas equal rates are expected for the latter [2].It has been suggested that both processes have played an important role in the rapid evolution of RNA interference-related pathways, including the long-and short-term evolutionary history of the Argonautes, the effectors of RNAi [3][4][5].The RNAi-related pathways comprise a range of small-RNA mechanisms best known for their roles in mediating the control of gene expression, antiviral responses, and defence against mobile genetic elements. Respectively, these include the miRNA pathway (Dicer-1 and Argonaute-1 in insects [6]), the siRNA pathway (Dcr-2 and Argounate 2 in insects [7]), and the piRNA pathway (piwi-family Argonaute AGO3 and Piwi/Aub in insects [8,9]).…”

“…This is best studied in Drosophila [19][20][21], but is also detectable in other invertebrates [22].It has been hypothesized that this may be a consequence of parasite-mediated 'arms-race' coevolution [20,23], either through conflict with parasite-encoded immune suppressors-as widely seen in RNA viruses [24]-or in the case of the piRNA pathway, through selection for 're-tuning' suppression mechanisms [25].Adaptive evolution of RNAi pathways is partly reflected in the gain, loss, and functional divergence of Argonaute-family duplications [26]. For example, within the Drosophilidae-an important model for RNAi-related pathways of animals-Piwi has been duplicated in the lineages leading to Phortica variegata and Scaptodrosophila deflexa [3], and Ago2 has been duplicated in those leading to S. deflexa, D. willistoni, D. melanogaster (where only one paralog remains -the canonical Ago2) and D.pseudoobscura [4]. This is particularly striking in the obscura group of Drosophila species [27], which has experienced at least 6 independent duplications of Ago2 over the last 20 million years, with all…”

“…It has been suggested that both processes have played an important role in the rapid evolution of RNA interference-related pathways, including the long-and short-term evolutionary history of the Argonautes, the effectors of RNAi [3][4][5].…”