Nematostella miRNAs

Vertebrates and insects display a significantly more complex body plan than basally branching metazoa such as Cnidaria or sponges. Yet, how morphological complexity is encoded is still an unsolved question. Since the gene repertoire of Cnidaria is surprisingly complex, it is now thought that gene interactions and gene regulation may play a decisive role in this regard. Recently, microRNAs (miRNAs) have been invoked in the evolutionary rise of morphological complexity from early metazoans to vertebrates and insects. miRNAs are RNAs of ~21-24 nucleotides with pivotal regulatory roles in diverse developmental and physiological pathways. They inhibit the translation of mRNAs and thus constitute a "tuning" system for controlling post-transcriptional expression. While the understanding of miRNAs function in bilaterians such as flies, nematodes and mammals is expanding rapidly, little is known about miRNAs in other animals, in particular morphologically simple and basal phyla. The starlet sea anemone, Nematostella vectensis is a rising model organism, which enables developmental biology studies in the early branching metazoan phylum Cnidaria under lab conditions. Recently, 40 miRNAs were found in N. vectensis but only one of them is a clear homologue of a bilaterian miRNA (miR-100). These 40 miRNAs result, however, of a pooled mixture of all early developemental stages, which makes it likely that temporally or spatially restricted miRNAs were missed. In order to unravel the full complexity of miRNAs and their role in post- transcriptional regulation in Nematostella we therefore first aim to identify the full complement of miRNAs by deep sequencing of 10 different developmental stages. Our unique culture system consisting of 500 tanks allows to provide the necessary material. Next, we intend to study the expression patterns and function of the miRNAs in Nematostella. Specifically, we will use in-situ hybridization, morpholino injection and transgenic manipulations to fulfill these aims. Once we have identified a miRNA whose knockdown results in a noticeable phenotype we will use various bioinformatic and experimental approaches to reveal and verify its target mRNAs. This research will potentially uncover the role of miRNA in a basal animal whose common ancestor with bilaterians lived ~600 million years ago and may shed light into the question, what the ancestral role of metazoan miRNAs was in a very plastic animal consisting of only few cell types.

The regulation of gene expression can occur at several different levels: first at the transcriptional level, when a given gene is transcribed from DNA into messenger RNA (mRNA), second at the post-transcriptional level, when the mRNA is translated into protein. Research of the last few years has revealed that the latter level is strongly controlled by small non-coding RNAs, called microRNAs (miRNAs), which bind in a sequence-specific manner to target mRNAs and inhibit translation or lead to a destabilization and decay of the mRNA. In humans, miRNAs have been implicated in a large diversity of developmental and physiological processes and their misregulation is often causative for diseases and abberrant development. miRNAs have been found in almost all animals and plants, but not in protists and fungi. The evolutionary origin of animal miRNAs is obscure, yet, they have been considered to have evolved independently of plant miRNAs, because their biogenesis is different, they do not share any sequence similarity and their mode of action is very different. In order to shed light into the evolution of miRNAs in early animals, we surveyed the role of miRNAs in a representative of an early evolving lineage, the cnidarians (e.g. sea anemones, corals, jellyfish). We first assessed the diversity of miRNAs by sequencing small RNAs from 8 developmental stages of the sea anemone Nematostella vectensis, including mature males and females. We found a total of 87 miRNAs, which is roughly in the order of magnitude of other, more complex animals. Yet, while animals as different as vertebrates and insects share over 30 miRNA families, only 1 out of the 87 sea anemone miRNAs is shared with other animals, indicating that cnidarians have a rather unique set of miRNAs. When we assessed the biogenesis machinery, we found that Nematostella has all necessary proteins typical of animal biogenesis, yet, also those proteins typical for plant biogenesis, several of which are not found in other animals. We next addressed the mode of action and found to our surprise that sea anemone miRNAs work by the same mechanism as plant miRNAs: they bind with almost perfect complementarity to their target mRNA and destroy it by slicing at a specific position. Taken together, our experimental and bioinformatic evidence strongly suggests that miRNAs evolved from a common ancestor with plants and only evolved the "animal"-type mechanism and biogenesis after divergence of the cnidarians. We speculate that this might have been a crucial step in the evolution of animal complexity.