Venom trade-off shapes interspecific interactions, physiology, and reproduction.

The ability of an animal to effectively capture prey and defend against predators is pivotal for survival. Venom is often a mixture of many components including toxin proteins that shape predator-prey interactions. Here, we used the sea anemone Nematostella vectensis to test the impact of toxin genotypes on predator-prey interactions. We developed a genetic manipulation technique to demonstrate that both transgenically deficient and a native Nematostella strain lacking a major neurotoxin (Nv1) have a reduced ability to defend themselves against grass shrimp, a native predator. In addition, secreted Nv1 can act indirectly in defense by attracting mummichog fish, which prey on grass shrimp. Here, we provide evidence at the molecular level of an animal-specific tritrophic interaction between a prey, its antagonist, and a predator. Last, this study reveals an evolutionary trade-off, as the reduction of Nv1 levels allows for faster growth and increased reproductive rates.

An ancient pan-cnidarian microRNA regulates stinging capsule biogenesis in Nematostella vectensis.

An ancient evolutionary innovation of a novel cell type, the stinging cell (cnidocyte), appeared >600 million years ago in the phylum Cnidaria (sea anemones, corals, hydroids, and jellyfish). A complex bursting nano-injector of venom, the cnidocyst, is embedded in cnidocytes and enables cnidarians to paralyze their prey and predators, contributing to this phylum's evolutionary success. In this work, we show that post-transcriptional regulation by a pan-cnidarian microRNA, miR-2022, is essential for biogenesis of these cells in the sea anemone Nematostella vectensis. By manipulation of miR-2022 levels in a transgenic reporter line of cnidocytes, followed by transcriptomics, single-cell data analysis, prey paralysis assays, and cell sorting of transgenic cnidocytes, we reveal that miR-2022 enables cnidocyte biogenesis in Nematostella, while exhibiting a conserved expression domain with its targets in cnidocytes of other cnidarian species. Thus, here we revealed a functional basis to the conservation of one of nature's most ancient microRNAs.

The Polycomb repressive complex 2 deposits H3K27me3 and represses transposable elements in a broad range of eukaryotes.

The mobility of transposable elements (TEs) contributes to evolution of genomes. Their uncontrolled activity causes genomic instability; therefore, expression of TEs is silenced by host genomes. TEs are marked with DNA and H3K9 methylation, which are associated with silencing in flowering plants, animals, and fungi. However, in distantly related groups of eukaryotes, TEs are marked by H3K27me3 deposited by the Polycomb repressive complex 2 (PRC2), an epigenetic mark associated with gene silencing in flowering plants and animals. The direct silencing of TEs by PRC2 has so far only been shown in one species of ciliates. To test if PRC2 silences TEs in a broader range of eukaryotes, we generated mutants with reduced PRC2 activity and analyzed the role of PRC2 in extant species along the lineage of Archaeplastida and in the diatom P. tricornutum. In this diatom and the red alga C. merolae, a greater proportion of TEs than genes were repressed by PRC2, whereas a greater proportion of genes than TEs were repressed by PRC2 in bryophytes. In flowering plants, TEs contained potential cis-elements recognized by transcription factors and associated with neighbor genes as transcriptional units repressed by PRC2. Thus, silencing of TEs by PRC2 is observed not only in Archaeplastida but also in diatoms and ciliates, suggesting that PRC2 deposited H3K27me3 to silence TEs in the last common ancestor of eukaryotes. We hypothesize that during the evolution of Archaeplastida, TE fragments marked with H3K27me3 were selected to shape transcriptional regulation, controlling networks of genes regulated by PRC2.

Some flies do not play ping-pong.

Genome integrity in animals depends on silencing of mobile elements by Piwi-interacting RNAs (piRNAs). A new study in this issue of PLOS Biology reveals recent evolutionary losses of key piRNA biogenesis factors in flies, highlighting adaptability by rapid shift to alternative piRNA biogenesis strategies.

Functional characterization of a 'plant-like' HYL1 homolog in the cnidarian Nematostella vectensis indicates a conserved involvement in microRNA biogenesis.

While the biogenesis of microRNAs (miRNAs) in both animals and plants depends on the RNase III Dicer, its partner proteins are considered distinct for each kingdom. Nevertheless, recent discovery of homologs of Hyponastic Leaves1 (HYL1), a 'plant-specific' Dicer partner, in the metazoan phylum Cnidaria, challenges the view that miRNAs evolved convergently in animals and plants. Here, we show that the HYL1 homolog Hyl1-like a (Hyl1La) is crucial for development and miRNA biogenesis in the cnidarian model Nematostella vectensis. Inhibition of Hyl1La by morpholinos resulted in metamorphosis arrest in Nematostella embryos and a significant reduction in levels of most miRNAs. Further, meta-analysis of morphants of miRNA biogenesis components, like Dicer1, shows clustering of their miRNA profiles with Hyl1La morphants. Strikingly, immunoprecipitation of Hyl1La followed by quantitative PCR revealed that in contrast to the plant HYL1, Hyl1La interacts only with precursor miRNAs and not with primary miRNAs. This was complemented by an in vitro binding assay of Hyl1La to synthetic precursor miRNA. Altogether, these results suggest that the last common ancestor of animals and plants carried a HYL1 homolog that took essential part in miRNA biogenesis and indicate early emergence of the miRNA system before plants and animals separated.

In both animals and plants, small molecules known as micro ribonucleic acids (or miRNAs for short) control the amount of proteins cells make from instructions encoded in their DNA. Cells make mature miRNA molecules by cutting and modifying newly-made RNA molecules in two stages. Some of the components animals and plants utilize to make and use miRNAs are similar, but most are completely different. For example, in plants an enzyme known as Dicer cuts newly made RNAs into mature miRNAs with the help of a protein called HYL1, whereas humans and other animals do not have HYL1 and Dicer works with alternative partner proteins, instead. Therefore, it is generally believed that miRNAs evolved separately in animals and plants after they split from a common ancestor around 1.6 billion years ago. Recent studies on sea anemones and other primitive animals challenge this idea. Proteins similar to HYL1 in plants have been discovered in sea anemones and sponges, and sea anemone miRNAs show several similarities to plant miRNAs including their mode of action. However, it is not clear whether these HYL1-like proteins work in the same way as their plant counterparts. Here, Tripathi, Admoni et al. investigated the role of the HYL1-like protein in sea anemones. The experiments found that this protein was essential for the sea anemones to make miRNAs and to grow and develop properly. Unlike HYL1 in plants ­ which is involved in both stages of processing newly-made miRNAs into mature miRNAs ­ the sea anemone HYL1-like protein only helped in the second stage to make mature miRNAs from intermediate molecules known as precursor miRNAs. These findings demonstrate that some of the components plants use to make miRNAs also perform similar roles in sea anemones. This suggests that the miRNA system evolved before the ancestors of plants and animals separated from each other. Questions for future studies will include investigating how plants and animals evolved different miRNA machinery, and why sponges and jellyfish have HYL1-like proteins, whereas humans and other more complex animals do not.

Conservation and turnover of miRNAs and their highly complementary targets in early branching animals.

MicroRNAs (miRNAs) are crucial post-transcriptional regulators that have been extensively studied in Bilateria, a group comprising the majority of extant animals, where more than 30 conserved miRNA families have been identified. By contrast, bilaterian miRNA targets are largely not conserved. Cnidaria is the sister group to Bilateria and thus provides a unique opportunity for comparative studies. Strikingly, like their plant counterparts, cnidarian miRNAs have been shown to predominantly have highly complementary targets leading to transcript cleavage by Argonaute proteins. Here, we assess the conservation of miRNAs and their targets by small RNA sequencing followed by miRNA target prediction in eight species of Anthozoa (sea anemones and corals), the earliest-branching cnidarian class. We uncover dozens of novel miRNAs but only a few conserved ones. Further, given their high complementarity, we were able to computationally identify miRNA targets in each species. Besides evidence for conservation of specific miRNA target sites, which are maintained between sea anemones and stony corals across 500 Myr of evolution, we also find indications for convergent evolution of target regulation by different miRNAs. Our data indicate that cnidarians have only few conserved miRNAs and corresponding targets, despite their high complementarity, suggesting a high evolutionary turnover.

Unravelling the developmental and functional significance of an ancient Argonaute duplication.

MicroRNAs (miRNAs) base-pair to messenger RNA targets and guide Argonaute proteins to mediate their silencing. This target regulation is considered crucial for animal physiology and development. However, this notion is based exclusively on studies in bilaterians, which comprise almost all lab model animals. To fill this phylogenetic gap, we characterize the functions of two Argonaute paralogs in the sea anemone Nematostella vectensis of the phylum Cnidaria, which is separated from bilaterians by ~600 million years. Using genetic manipulations, Argonaute-immunoprecipitations and high-throughput sequencing, we provide experimental evidence for the developmental importance of miRNAs in a non-bilaterian animal. Additionally, we uncover unexpected differential distribution of distinct miRNAs between the two Argonautes and the ability of one of them to load additional types of small RNAs. This enables us to postulate a novel model for evolution of miRNA precursors in sea anemones and their relatives, revealing alternative trajectories for metazoan miRNA evolution.

Too Many False Targets for MicroRNAs: Challenges and Pitfalls in Prediction of miRNA Targets and Their Gene Ontology in Model and Non-model Organisms.

Short ("seed") or extended base pairing between microRNAs (miRNAs) and their target RNAs enables post-transcriptional silencing in many organisms. These interactions allow the computational prediction of potential targets. In model organisms, predicted targets are frequently validated experimentally; hence meaningful miRNA-regulated processes are reported. However, in non-models, these reports mostly rely on computational prediction alone. Many times, further bioinformatic analyses such as Gene Ontology (GO) enrichment are based on these in silico projections. Here such approaches are reviewed, their caveats are highlighted and the ease of picking false targets from predicted lists is demonstrated. Discoveries that shed new light on how miRNAs evolved to regulate targets in various phyletic groups are discussed, in addition to the pitfalls of target identification in non-model organisms. The goal is to prevent the misuse of bioinformatic tools, as they cannot bypass the biological understanding of miRNA-target regulation.

Cell type-specific expression profiling unravels the development and evolution of stinging cells in sea anemone.

BACKGROUND: Cnidocytes are specialized cells that define the phylum Cnidaria. They possess an "explosive" organelle called cnidocyst that is important for prey capture and anti-predator defense. An extraordinary morphological and functional complexity of the cnidocysts has inspired numerous studies to investigate their structure and development. However, the transcriptomes of the cells bearing these unique organelles are yet to be characterized, impeding our understanding of the genetic basis of their biogenesis. RESULTS: In this study, we generated a nematocyte reporter transgenic line of the sea anemone Nematostella vectensis using the CRISPR/Cas9 system. By using a fluorescence-activated cell sorter (FACS), we have characterized cell type-specific transcriptomic profiles of various stages of cnidocyte maturation and showed that nematogenesis (the formation of functional cnidocysts) is underpinned by dramatic shifts in the spatiotemporal gene expression. Among the genes identified as upregulated in cnidocytes were Cnido-Jun and Cnido-Fos1-cnidarian-specific paralogs of the highly conserved c-Jun and c-Fos proteins of the stress-induced AP-1 transcriptional complex. The knockdown of the cnidocyte-specific c-Jun homolog by microinjection of morpholino antisense oligomer results in disruption of normal nematogenesis. CONCLUSIONS: Here, we show that the majority of upregulated genes and enriched biochemical pathways specific to cnidocytes are uncharacterized, emphasizing the need for further functional research on nematogenesis. The recruitment of the metazoan stress-related transcription factor c-Fos/c-Jun complex into nematogenesis highlights the evolutionary ingenuity and novelty associated with the formation of these highly complex, enigmatic, and phyletically unique organelles. Thus, we provide novel insights into the biology, development, and evolution of cnidocytes.

The methyltransferase HEN1 is required in Nematostella vectensis for microRNA and piRNA stability as well as larval metamorphosis.

Small non-coding RNAs (sRNAs) such as microRNAs (miRNAs), small interfering RNAs (siRNAs) and piwi-interacting RNAs (piRNAs) regulate the levels of endogenous, viral and transposable element RNA in plants (excluding piRNAs) and animals. These pathways are explored mainly in bilaterian animals, such as vertebrates, arthropods and nematodes, where siRNAs and piRNAs, but not miRNAs bind their targets with a perfect match and mediate the cleavage of the target RNA. Methylation of the 3' ends of piRNAs and siRNAs by the methyltransferase HEN1 protects these sRNAs from degradation. There is a noticeable selection in bilaterian animals against miRNA-mRNA perfect matching, as it leads to the degradation of miRNAs. Cnidarians (sea anemones, corals, hydroids and jellyfish), are separated from bilaterians by more than 600 million years. As opposed to bilaterians, cnidarian miRNAs frequently bind their targets with a nearly perfect match. Knowing that an ortholog of HEN1 is widely expressed in the sea anemone Nematostella vectensis, we tested in this work whether it mediates the stabilization of its sRNAs. We show that the knockdown of HEN1 in Nematostella results in a developmental arrest. Small RNA sequencing revealed that the levels of both miRNAs and piRNAs drop dramatically in the morphant animals. Moreover, knockdown experiments of Nematostella Dicer1 and PIWI2, homologs of major bilaterian biogenesis components of miRNAs and piRNAs, respectively, resulted in developmental arrest similar to HEN1 morphants. Our findings suggest that HEN1 mediated methylation of sRNAs reflects the ancestral state, where miRNAs were also methylated. Thus, we provide the first evidence of a methylation mechanism that stabilizes miRNAs in animals, and highlight the importance of post-transcriptional regulation in non-bilaterian animals.

Dynamics of venom composition across a complex life cycle.

Little is known about venom in young developmental stages of animals. The appearance of toxins and stinging cells during early embryonic stages in the sea anemone Nematostella vectensis suggests that venom is already expressed in eggs and larvae of this species. Here, we harness transcriptomic, biochemical and transgenic tools to study venom production dynamics in Nematostella. We find that venom composition and arsenal of toxin-producing cells change dramatically between developmental stages of this species. These findings can be explained by the vastly different interspecific interactions of each life stage, as individuals develop from a miniature non-feeding mobile planula to a larger sessile polyp that predates on other animals and interact differently with predators. Indeed, behavioral assays involving prey, predators and Nematostella are consistent with this hypothesis. Further, the results of this work suggest a much wider and dynamic venom landscape than initially appreciated in animals with a complex life cycle.