Final Report Summary - NEMATOSTELLAMICRORNA (Functional study of MicroRNAs in the starlet sea anemone Nematostella vectensis (Cnidaria; Anthozoa)) MicroRNAs (miRNAs) are short nucleic acids that play important part in development and physiology of animals and plants as they regulate the expression of many genes by binding to messenger RNAs (mRNAs), the gene products that encode for proteins. In Bilateria, which comprise most of extant animals such as vertebrates, insects and nematodes, miRNAs regulate messenger RNAs via base-pairing (specific recognition of two nucleic acids) of a short sub-sequence (the miRNA “seed”) to their target, subsequently promoting inhibition of protein translation and mRNA instability. In contrast, in plants many miRNAs guide direct cleavage of highly complementary mRNA targets by binding to them throughout their entire length. As little is known about miRNA function in non-bilaterian animals, we investigated the repertoire and biological activity of miRNAs in the sea anemone Nematostella vectensis, a representative of Cnidaria, the sister phylum of Bilateria. Our work uncovered tens of novel miRNAs in Nematostella, increasing the total miRNA gene count to 88, which is comparable to the number of miRNAs found in some bilaterians such as flatworms and segmented worms. This finding negates previous theories suggesting that a main reason for the higher body plan complexity of Bilateria is due to a higher number of miRNAs found in the genomes of this animal group. However, only a handful of the Nematostella miRNAs we detected seem to be conserved in other cnidarians such as corals and hydras and only a single miRNA is shared between Nematostella and Bilateria. This suggests that the rate of birth and death of miRNA genes in Cnidaria might be much higher than in other animal groups. We further showed by biochemical and computational means that in Nematostella, miRNAs frequently direct the cleavage of their mRNA targets via nearly perfect complementarity extending beyond the seed. This mode of action is reminiscent of that of small interfering RNAs and plant miRNAs. It appears to be common to Cnidaria, as several of the miRNA target sites are conserved among distantly related sea anemone species and since we also detected miRNA-directed cleavage in the far-related cnidarian Hydra magnipapillata, which is separated from Nematostella by more than 500 million years. Interestingly, many of the cleaved miRNA targets we detected in Nematostella are similar to important bilaterian genes involved in body development and cell identity and include genes such as HoxD, Six3/6 and Vasa2. The identification of these targets strongly suggests that the miRNA pathway plays an important role in regulating development in Cnidaria, similar to the situation in bilaterian animals and in plants. However, unlike in bilaterian animals, Nematostella miRNAs are commonly co-expressed with their target transcripts, resulting in a regulatory topology rare for miRNAs and known as incoherent feed-forward loop. In light of this observed topology and because of the common cleavage of miRNA targets in Nematostella, we propose that post-transcriptional regulation by miRNAs might function very differently in Cnidaria and Bilateria. Furthermore, the similar mode of action of miRNAs in Cnidaria and plants suggests that this is an ancestral state that might have existed in the common ancestor of plants and animals. This view is contrasted to the current scientific view that supports independent evolvement of the miRNA pathways of plants and animals. In summary, our project revealed important differences in the regulation of expression by miRNAs in Cnidaria compared to Bilateria and raised the possibility that this important pathway is much older than previously thought. Our findings also highlight the importance of studying non-bilaterian model animals as a comparison to bilaterian animals and that evolutionary hypotheses can dramatically change when neglected groups of organisms are studied.