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. 2018 Nov 1;18(1):160.
doi: 10.1186/s12862-018-1274-2.

Diverse RNA interference strategies in early-branching metazoans

Andrew D Calcino  1   2 , Selene L Fernandez-Valverde  1   3 , Ryan J Taft  4   5 , Bernard M Degnan  6
Affiliations

Diverse RNA interference strategies in early-branching metazoans

Andrew D Calcino et al. BMC Evol Biol. .

Abstract

Background: Micro RNAs (miRNAs) and piwi interacting RNAs (piRNAs), along with the more ancient eukaryotic endogenous small interfering RNAs (endo-siRNAs) constitute the principal components of the RNA interference (RNAi) repertoire of most animals. RNAi in non-bilaterians - sponges, ctenophores, placozoans and cnidarians - appears to be more diverse than that of bilaterians, and includes structurally variable miRNAs in sponges, an enormous number of piRNAs in cnidarians and the absence of miRNAs in ctenophores and placozoans.

Results: Here we identify thousands of endo-siRNAs and piRNAs from the sponge Amphimedon queenslandica, the ctenophore Mnemiopsis leidyi and the cnidarian Nematostella vectensis using a computational approach that clusters mapped small RNA sequences and annotates each cluster based on the read length and relative abundance of the constituent reads. This approach was validated on 11 small RNA libraries in Drosophila melanogaster, demonstrating the successful annotation of RNAi-associated loci with properties consistent with previous reports. In the non-bilaterians we uncover seven new miRNAs from Amphimedon and four from Nematostella as well as sub-populations of candidate cis-natural antisense transcript (cis-NAT) endo-siRNAs. We confirmed the absence of miRNAs in Mnemiopsis but detected an abundance of endo-siRNAs in this ctenophore. Analysis of putative piRNA structure suggests that conserved localised secondary structures in primary transcripts may be important for the production of mature piRNAs in Amphimedon and Nematostella, as is also the case for endo-siRNAs.

Conclusion: Together, these findings suggest that the last common ancestor of extant animals did not have the entrained RNAi system that typifies bilaterians. Instead it appears that bilaterians, cnidarians, ctenophores and sponges express unique repertoires and combinations of miRNAs, piRNAs and endo-siRNAs.

Keywords: Cnidarian; Ctenophore; Demosponge; Endo-siRNA; Non-bilaterian; RNAi; miRNA; piRNA.

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Conflict of interest statement

Ethics approval and consent to participate

No specific ethics approval was required for this project.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Authors' Note A paper was published after acceptance of this manuscript providing evidence for animal-like microRNAs and the miRNA biogenesis machinery in the unicellular ichthyosporeans (Bråte J, Neumann RS, Fromm B, Haraldsen AAB, Tarver, JE., Suga H, et al. (2018). Unicellular Origin of the Animal MicroRNA Machinery. Current Biology, 10.1016/j.cub.2018.08.018). Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Genomic context of endo-siRNA and piRNA cluster expression for unique and multi-mapping clusters. Each colour-coded segment represents the percentage of endo-siRNA or piRNA clusters mapping to the specified genomic elements. Percentages slightly exceed 100% due to some regions of the genome encoding multiple types of element. The genome column shows the percentage of the genome covered by the specified genomic elements. For Drosophila, d1) 12–24 h embryo, d2) first instar larvae 1, d3) first instar larvae 2, d4) third instar larvae 1, d5) third instar larvae 2, d6) 0–1 day pupae, d7) 2–4 day pupae 1, d8) 2–4 day pupae 2, d9) male adult body, d10) female adult body, d11) female adult head; Amphimedon a1) pre-competent larvae, a2) competent larvae, a3) juvenile, a4) adult; Mnemiopsis, m1) Mnemiopsis 1, m2) Mnemiopsis 2; Nematostella, n1) unfertilized eggs, n2) blastula, n3) gastrula, n4) early planula larvae, n5) late planula larvae, n6) metamorphosing, n7) primary polyp, n8) male adult, n9) female adult
Fig. 2
Fig. 2
Randfold results for endo-siRNA and piRNA clusters. Each bar represents the percentage of clusters with Randfold p-values equal to or less than the values stated on the X-axis. The more stringent the p-value cutoff, the more confidence there is that the secondary structure of the native sequence is more stable than a randomised version of itself. For each graph, the Randfold scores of either endo-siRNAs or piRNAs are compared to the Randfold scores of all clusters not annotated as endo-siRNAs or piRNAs. For each species, all available datasets were pooled
Fig. 3
Fig. 3
Library contributions from each RNAi component as a percentage of total library depth. Total contributions of miRNAs, endo-siRNAs, piRNAs and Mnemiopsis 25-mer clusters to total library depth. For each, only a single copy of each multi-mapping read was considered
Fig. 4
Fig. 4
Co-expression of uniquely-mapping endo-siRNA and piRNA clusters. Each plot is divided in to groups of coloured scaffolds/chromosomes, each of which represents a developmental stage; four stages in Amphimedon, nine stages in Nematostella and 11 stages in Drosophila. For each plot, the earliest developmental stage is marked with an arrow indicating the chronological order of developmental stages. Links between scaffolds/chromosomes indicate co-expression from a particular endo-siRNA or piRNA cluster in the two linked developmental stages. For Drosophila, all chromosomes are represented while for Amphimedon and Nematostella, the ten largest genomic scaffolds were used. Beginning with the developmental stage indicated by the arrow, the stages for Amphimedon, Nematostella and Drosophila are as per Fig. 1. For each species, the links shared with a single developmental stage are coloured black for emphasis while the rest are coloured grey. For Amphimedon the emphasised stage is the pre-competent larvae (a1), for Nematostella the female adult (n9) and for Drosophila, the female adult head (d11)
Fig. 5
Fig. 5
Characterisation of Mnemiopsis 25-mer clusters. The Mnemiopsis 25-mer clusters were annotated using the same methods employed for characterisation of the three known RNAi classes. a Read length distribution of all mapped sRNAs from the Woods Hole, MA, USA library (Mnemiopsis 1) and the Miami, FL, USA library (Mnemiopsis 2). Distinct reads (red) and total read counts (blue) of all mapped sRNA size classes reveals peaks of mapped sRNAs at 21 and 25 nt in both libraries. b Nucleotide biases along the length of all sRNAs mapping to 25-mer clusters. sRNAs were anchored at their 5′ nucleotide and biases are displayed as a percentage of the contribution of each nucleotide at each position. Of note is the tendency for a uracil at position 1. c Genomic context of 25-mer cluster expression (as per Fig. 1) demonstrates the lack of enrichment of 25-mer clusters from coding genes or transposons. d Randfold results (as per Fig. 2) demonstrate a lack of evidence for secondary structure in 25-mer clusters
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