Non-coding RNA in the evolution of Animals

The goal of this project has been to understand how animals originated and evolved from their single-celled ancestors. Particularly, we wanted to investigate the role of so-called non-protein coding RNAs in this evolutionary event. The origin of animals was a giant evolutionary leap from the morphologically simpler organisms known as single-celled holozoans (Holozoa is the term for animals and their single-celled relatives). Suddenly, a single cell would give rise to a whole body with different cell types and morphologies characterized by unique patterns of gene expression. One hypothesis is that this transition was made possible by inventions in the way genes are regulated, their timing and combination, and not because of inventions of new genes with new abilities. Non-protein coding RNAs (ncRNAs) had been known for a long time, but had seen a renewed interest right before we started this project. Mainly this was due to developments in gene sequencing which had revealed an enormous amount of RNA with unknown functions. And a few studies on animals had even shown that some ncRNAs had were essential in regulating how other genes are used. But it was not known whether such ncRNAs arose late in the evolution of animals, and thereby a signature of higher animals, or whether they were present at the very origin of animals. A main aim of this project has therefore been to identify and characterize ncRNAs in the earliest evolving animal lineages and their closest single-celled relatives. We have also tried to understand the function of these ncRNAs and the significance of ncRNAs in the early evolution of animals. Much of our attention has been devoted to a special type of ncRNA called microRNAs (miRNAs). These are short RNA molecules that regulate how other genes are used. And they serve a very important function by controlling the correct development of both plants and animals. In animals, miRNAs are processed by a special protein machinery called the Microprocessor. And miRNAs cannot exist without these proteins. When we started this project, the Microprocessor had not been found outside animals and was believed to be an animal innovation. Exactly when this innovation had taken place was not known, but it had to have happened very early in animal evolution. Perhaps as early as in the last common ancestor of animals. But we have discovered that actually the Microprocessor proteins, as well as the miRNAs, are present in a group of single-celled holozoans called Ichthyosporea. Ichthyosporeans are closely related to both animals and fungi. And what is interesting with these species is that they have the ability to form partially multicellular structures, a feature believed to be a preadaptation to animal multicellularity. Could the presence of gene regulatory miRNAs be related to the ability to form complex morphological structures? In any case, this shows that both the Microprocessor and the miRNAs were not invented early in animal evolution. This work is openly available in the journal Current Biology (doi: 10.1016/j.cub.2018.08.018). We have followed this up with experiments aiming at revealing the function of these miRNAs. We did a so-called ?degradome sequencing? (where we can find gene products which have been cleaved in the cell), which showed that the miRNAs probably binds to gene targets and lead to cleavage and down regulation. If this is confirmed it will be a function more similar to that of plant miRNAs, and not the normal animal miRNA function. Men flere forsøk gjenstår for å bekrefte dette. This experiment was performed as part of the master thesis of Hengyi Zhu in 2019 (http://urn.nb.no/URN:NBN:no-71761). In addition to miRNAs, we have also studied another group of ncRNAs called long non-coding RNAs (lncRNAs). This work has been done by two master students, Line Røsæg and Andreas Evenstad who both finished their theses in 2017 (http://urn.nb.no/URN:NBN:no-57753 and http://urn.nb.no/URN:NBN:no-57751). Line and Andreas studied a very strange group of animals called comb jellies, which were among the first animals to evolve. They found out that lncRNAs are highly active during the development from larvae to adults. In addition, Andreas showed that the same lncRNAs that were active during development were also highly conserved during evolution of comb jellies. We have also followed up this work on lncRNAs by investigating whether they also exist in the same group of single-celled eukaryotes where we found the miRNAs, the ichthyosporeans. A PhD student on the project, Arthur Haraldsen had a research stay for 3 months in the group of Iñaki Ruiz-Trillo in Barcelona to sequence the transcriptome during the entire life cycle. And similar to the comb jellies we found that a lot of evolutionary conserved lncRNAs had a dynamically regulated gene expression during cell development. This work is published in the journal eLife (doi: 10.7554/eLife.49801).

The results of the project have shed new light on our understanding of the evolution of animals. We have strengthened emerging views that the single-celled ancestors of animals were probably genetically highly complex, and had a cell biology and lifestyle that rivals their animal counterparts. This changes our view of the evolution of how animals evolved, and that this perhaps did not require a massive innovation of genes and genetic capabilities after all. Importantly, the project has opened up a new area of research. We have shown that regulatory RNAs, and in particular microRNAs, are present in the single-celled relatives of animals. And although we have caught a glimpse of their function, we still know very little about these regulatory elements and further research is therefore needed.

The overall goal of the project is to understand how animals originated and evolved. The project is unique in that it emphasizes the role of gene regulation by non-coding RNAs (ncRNAs) in the transition from unicellular eukaryotes to multicellular animals. The origin of animals was a giant evolutionary leap from relatively simple single celled Choanozoa. Suddenly, a single genotype would give rise to different cell types and morphologies characterized by unique patterns of gene expression. It is believed that this leap was made possible by inventions in the gene regulatory machinery, and that the driver of morphological evolution in animals is mostly changes in gene regulation and not the invention of new genes. In higher animals gene regulatory non-coding RNAs have been found to play important roles in developmental processes. But so far, it is not known whether these are inventions that took place late in animal evolution or whether ncRNAs were present already in the last common ancestor of animals, or even earlier. The aim of this project is therefore to identify and characterize the ncRNA repertoire of basal animals and their closest ancestors, Choanozoa. Further, it aims to understand the function of these ncRNAs and the evolutionary significance of ncRNAs in the evolution of animals. This proposal is aimed to the call for Young Research Talents. I, the applicant, am currently the project leader of a personal post doc project on non-coding RNAs in evolution. The proposed project will give me the opportunity to establish a career as an independent researcher in the intersection of evolutionary developmental biology and molecular genetics.