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.

Functional Characterization of the Cnidarian Antiviral Immune Response Reveals Ancestral Complexity.

Animals evolved a broad repertoire of innate immune sensors and downstream effector cascades for defense against RNA viruses. Yet, this system varies greatly among different bilaterian animals, masking its ancestral state. In this study, we aimed to characterize the antiviral immune response of the cnidarian Nematostella vectensis and decipher the function of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) known to detect viral double-stranded RNA (dsRNA) in bilaterians but activate different antiviral pathways in vertebrates and nematodes. We show that polyinosinic:polycytidylic acid (poly(I:C)), a mimic of long viral dsRNA and a primary ligand for the vertebrate RLR melanoma differentiation-associated protein 5 (MDA5), triggers a complex antiviral immune response bearing features distinctive for both vertebrate and invertebrate systems. Importantly, a well-characterized agonist of the vertebrate RIG-I receptor does not induce a significant transcriptomic response that bears signature of the antiviral immune response, which experimentally supports the results of a phylogenetic analysis indicating clustering of the two N. vectensis RLR paralogs (NveRLRa and NveRLRb) with MDA5. Furthermore, the results of affinity assays reveal that NveRLRb binds poly(I:C) and long dsRNA and its knockdown impairs the expression of putative downstream effector genes including RNA interference components. Our study provides for the first time the functional evidence for the conserved role of RLRs in initiating immune response to dsRNA that originated before the cnidarian-bilaterian split and lay a strong foundation for future research on the evolution of the immune responses to RNA viruses.

TATA Binding Protein (TBP) Promoter Drives Ubiquitous Expression of Marker Transgene in the Adult Sea Anemone Nematostella vectensis.

Nematostella vectensis has emerged as one as the most established models of the phylum Cnidaria (sea anemones, corals, hydroids and jellyfish) for studying animal evolution. The availability of a reference genome and the relative ease of culturing and genetically manipulating this organism make it an attractive model for addressing questions regarding the evolution of venom, development, regeneration and other interesting understudied questions. We and others have previously reported the use of tissue-specific promoters for investigating the function of a tissue or a cell type of interest in vivo. However, to our knowledge, genetic regulators at the whole organism level have not been reported yet. Here we report the identification and utilization of a ubiquitous promoter to drive a wide and robust expression of the fluorescent protein mCherry. We generated animals containing a TATA binding protein (TBP) promoter upstream of the mCherry gene. Flow cytometry and fluorescent microscopy revealed expression of mCherry in diverse cell types, accounting for more than 90% of adult animal cells. Furthermore, we detected a stable mCherry expression at different life stages and throughout generations. This tool will expand the existing experimental toolbox to facilitate genetic engineering and functional studies at the whole organism level.