Metagenomic sequencing suggests a diversity of RNA interference-like responses to viruses across multicellular eukaryotes.

FM Waldron, GN Stone, DJ Obbard

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70 Citations (Scopus)


RNA interference (RNAi)-related pathways target viruses and transposable element (TE) transcripts in plants, fungi, and ecdysozoans (nematodes and arthropods), giving protection against infection and transmission. In each case, this produces abundant TE and virus-derived 20-30nt small RNAs, which provide a characteristic signature of RNAi-mediated defence. The broad phylogenetic distribution of the Argonaute and Dicer-family genes that mediate these pathways suggests that defensive RNAi is ancient, and probably shared by most animal (metazoan) phyla. Indeed, while vertebrates had been thought an exception, it has recently been argued that mammals also possess an antiviral RNAi pathway, although its immunological relevance is currently uncertain and the viral small RNAs (viRNAs) are not easily detectable. Here we use a metagenomic approach to test for the presence of viRNAs in five species from divergent animal phyla (Porifera, Cnidaria, Echinodermata, Mollusca, and Annelida), and in a brown alga—which represents an independent origin of multicellularity from plants, fungi, and animals. We use metagenomic RNA sequencing to identify around 80 virus-like contigs in these lineages, and small RNA sequencing to identify viRNAs derived from those viruses. We identified 21U small RNAs derived from an RNA virus in the brown alga, reminiscent of plant and fungal viRNAs, despite the deep divergence between these lineages. However, contrary to our expectations, we were unable to identify canonical (i.e. Drosophila- or nematode-like) viRNAs in any of the animals, despite the widespread presence of abundant micro-RNAs, and somatic transposon-derived piwi-interacting RNAs. We did identify a distinctive group of small RNAs derived from RNA viruses in the mollusc. However, unlike ecdysozoan viRNAs, these had a piRNA-like length distribution but lacked key signatures of piRNA biogenesis. We also identified primary piRNAs derived from putatively endogenous copies of DNA viruses in the cnidarian and the echinoderm, and an endogenous RNA virus in the mollusc. The absence of canonical virus-derived small RNAs from our samples may suggest that the majority of animal phyla lack an antiviral RNAi response. Alternatively, these phyla could possess an antiviral RNAi response resembling that reported for vertebrates, with cryptic viRNAs not detectable through simple metagenomic sequencing of wild-type individuals. In either case, our findings show that the antiviral RNAi responses of arthropods and nematodes, which are highly divergent from each other and from that of plants and fungi, are also highly diverged from the most likely ancestral metazoan state.
Original languageEnglish
Article numbere1007533
JournalPLoS Genetics
Issue number7
Publication statusPublished - 30 Jul 2018

Bibliographical note

Funding: This work was funded by a Leverhulme Trust grant to DJO, GNS and FMW (RPG-2013-168;, and work in DJO’s lab was partly supported by a Wellcome Trust strategic award to the Centre for Immunity, Infection and Evolution (WT095831; The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability Statement

Raw reads from RNAseq and small RNA sequencing are available from the NCBI Sequence Read Archive under BioProject accession PRJNA394213. Virus sequences (accession numbers MF189971-MF190055), and host RNAi gene sequences (accession numbers MF288049-MF288076), are available through GenBank. For each of the six species pools, the raw meta-transcriptomic contigs generated by Trinity are provided in compressed (gzipped) fasta format as unannotated contigs through the figshare repository doi:10.6084/m9.figshare.6803885.v1 (


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