Analysis of Hydra PIWI proteins and piRNAs uncover early evolutionary origins of the piRNA pathway

Abstract

To preserve genome integrity, an evolutionarily conserved small RNA-based silencing mechanism involving PIWI proteins and PIWI-interacting RNAs (piRNAs) represses potentially deleterious transposons in animals. Although there has been extensive research into PIWI proteins in bilaterians, these proteins remain to be examined in ancient phyla. Here, we investigated the PIWI proteins Hywi and Hyli in the cnidarian Hydra, and found that both PIWI proteins are enriched in multipotent stem cells, germline stem cells, and in the female germline. Hywi and Hyli localize to the nuage, a perinuclear organelle that has been implicated in piRNA-mediated transposon silencing, together with other conserved nuage and piRNA pathway components. Our findings provide the first report of nuage protein localization patterns in a non-bilaterian. Hydra PIWI proteins possess symmetrical dimethylarginines: modified residues that are known to aid in PIWI protein localization to the nuage and proper piRNA loading. piRNA profiling suggests that transposons are the major targets of the piRNA pathway in Hydra. Our data suggest that piRNA biogenesis through the ping-pong amplification cycle occurs in Hydra and that Hywi and Hyli are likely to preferentially bind primary and secondary piRNAs, respectively. Presumptive piRNA clusters are unidirectionally transcribed and primarily give rise to piRNAs that are antisense to transposons. These results indicate that various conserved features of PIWI proteins, the piRNA pathway, and their associations with the nuage were likely established before the evolution of bilaterians.

Characterization of the piRNA pathway during development of the sea anemone Nematostella vectensis

“…Hence, Nematostella piRNAs and their partner proteins possibly silence TEs in the soma in addition to the germline. Interestingly, the fact that piRNAs and piRNA-related proteins are also highly and widely expressed in Hydra, 25,33 and that piRNAs constitute the highest fraction of small RNAs in the sponge Amphimedon queenslandica 34 as well, suggest that these aspects represent shared traits of early-branching metazoans.…”

“…23,24 Maelstrom is fundamental for transcriptional transposon silencing and conserved in Hydra, Drosophila and mouse. 4,25,26 For comparison, we also included genes that encode microRNA machinery proteins (AGO1, AGO2, Dcr1, Dcr2 and GW182) in our analysis. According to their temporal expression patterns across developmental stages, the investigated genes cluster into three groups ( Fig.…”

“…A similar observation was made in Hydra: immunoprecipitation experiments against Hywi and Hyli detected physical association of these PIWI proteins and piRNAs mainly with transposon sequences but also, to a smaller extent, with protein-coding genes. 25,33 Approximately 5% and 12% of total and Hywi-bound piRNAs mapped to transcripts of protein-coding genes respectively. 25 To investigate the overlap of protein-coding genes as piRNA targets between Hydra and Nematostella, we performed a reciprocal BLAST analysis and found that no transcripts targeted by piRNAs are shared in these two species.…”

“…25,33 Approximately 5% and 12% of total and Hywi-bound piRNAs mapped to transcripts of protein-coding genes respectively. 25 To investigate the overlap of protein-coding genes as piRNA targets between Hydra and Nematostella, we performed a reciprocal BLAST analysis and found that no transcripts targeted by piRNAs are shared in these two species.…”

“…Recently, piRNAs and PIWI proteins were identified in the cnidarian Hydra. [14][15][16] Hywi and Hyli (the two paralogous Hydra PIWI proteins) are predominantly expressed in the germline as well as in the interstitial cell lineage (the so-called i-cells) including multipotent progenitor stem cells and germline stem cells, but also in epithelial stem cells. Hydra piRNAs comprise a large fraction among their entire repertoire of small RNAs and map to transposons.…”