Abstract
Platyhelminthes comprise one of the major phyla of invertebrate animals, inhabiting a wide range of ecosystems, and one of the most successful in adapting to parasitic life. Small non-coding RNAs have been implicated in regulating complex developmental transitions in model parasitic species. Notably, parasitic flatworms have lost Piwi RNA pathways but gained a novel Argonaute gene. Herein, we analyzed, contrasted and compared the conservation of small RNA pathways among several free-living species (a paraphyletic group traditionally known as ‘turbellarians’) and parasitic species (organized in the monophyletic clade Neodermata) to disentangle possible adaptations during the transition to parasitism. Our findings showed that complete miRNA and RNAi pathways are present in all analyzed free-living flatworms. Remarkably, whilst all ‘turbellarians’ have Piwi proteins, these were lost in parasitic Neodermantans. Moreover, two clusters of Piwi class Argonaute genes are present in all ‘turbellarians’. Interestingly, we identified a divergent Piwi class Argonaute in free living flatworms exclusively, which we named ‘Fliwi’. In addition, other key proteins of the Piwi pathways were conserved in ‘turbellarians’, while none of them were detected in Neodermatans. Besides Piwi and the canonical Argonaute proteins, a flatworm-specific class of Argonautes (FL-Ago) was identified in the analyzed species confirming its ancestrallity to all Platyhelminthes. Remarkably, this clade was expanded in parasitic Neodermatans, but not in free-living species. These phyla-specific Argonautes showed lower sequence conservation compared to other Argonaute proteins, suggesting that they might have been subjected to high evolutionary rates. However, key residues involved in the interaction with the small RNA and mRNA cleavage in the canonical Argonautes were more conserved in the FL-Agos than in the Piwi Argonautes. Whether this is related to specialized functions and adaptations to parasitism in Neodermatans remains unclear. In conclusion, differences detected in gene conservation, sequence and structure of the Argonaute family suggest tentative biological and evolutionary diversifications that are unique to Platyhelminthes. The remarkable divergencies in the small RNA pathways between free-living and parasitic flatworms indicate that they may have been involved in the adaptation to parasitism of Neodermatans.
Highlights
At the beginning of this century an unexpected and complex ‘RNA world’ started to be unveiled (Guil and Esteller, 2015), resulting in the discovery of novel layers of fine-tuned mechanisms for gene expression regulation, unimaginable until
While miRNA and short-interfering RNAs (siRNAs) Pathways Are Conserved Across Platyhelminths, the Complete Piwi Pathway Is Lost in Parasitic Flatworms
We provide a strong bioinformatic support for the presence and absence of key proteins involved in small RNA pathways in early diverging free-living flatworms, suggesting that miRNA regulation, piRNA mediated silencing and RNA interference (RNAi) are ancestral regulatory mechanisms in flatworms
Summary
At the beginning of this century an unexpected and complex ‘RNA world’ started to be unveiled (Guil and Esteller, 2015), resulting in the discovery of novel layers of fine-tuned mechanisms for gene expression regulation, unimaginable until . Regulatory activities were assigned to a growing range of new species of non-coding RNAs (Guil and Esteller, 2015). Single stranded non-coding RNAs of 20 to 30 nucleotides long are key mediators in small RNA pathways that underlie diverse biological processes. Piwi-RNAs (piRNAs) are small non-coding RNAs involved in the maintenance of genome stability by silencing transposable elements (TE) in germline cells (Weick and Miska, 2014). The presence of dsRNA molecules in the cell cytoplasm triggers a post-transcriptional degradation of complementary mRNA molecules. This pathway has been exploited as a reverse genetic tool to silence specific genes (Han, 2018). More than two decades after it was first applied to a flatworm species (Sánchez Alvarado and Newmark, 1999), it is still the main tool to study gene function in worms (Mourão et al, 2012; Wang et al, 2020)
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