Role of small non-coding RNA in fish development: comparative study on regulatory mechanisms

The major purpose of this project is to have an insight into evolution, biogenesis and functions of various regulatory non-coding RNA (ncRNA) in fish development. We have analysed ncRNA transcriptome in fish representing distant phylogenetic lineages, including representatives of 4 out of 5 superorders of Teleostei (zebrafish, Atlantic salmon, Atlantic cod, and Atlantic halibut), one Chondrostei (Siberian sturgeon), and one Holostei (spotted gar), being an outgroup to both Telostei and Chondrostei in term of whole genome duplication events. We focused on parental regulation of early developmental processes, and their switch to the zygotic regulation. We have profiled microRNA throughout the development and discovered novel microRNAs in the investigated species. We have demonstrated that the early embryonic thermal experience has a long-term effect on microRNA expression in juvenile Atlantic cod. We have characterized small RNA transcriptome during gonadal development in zebrafish. We are in process of characterization of piRNAs in the germline and early embryonic development. We performed functional validation of miRNA and their targets in gonadal development. The results indicate that small regulatory RNAs are massively present during the earliest developmental stages of distant fish species, and their dynamics is rapidly changing during the development. Their transcriptional units are active before massive maternal-to-zygotic transition, and they are functionally active in the earliest developmental stages.

We will investigate the distribution, evolutionary conservation, and properties of small, non-protein coding RNAs (ncRNAs), particularly microRNAs (miRNAs), which are important regulatory components of developmental processes and have biological roles and mechanisms of action that are to a great extent as yet undiscovered. Our approach involves a combination of computational and functional studies, including high-throughput sequencing and cutting edge functional genomics tools. We will involve national st ate-of-the art infrastructure resources (SOLiD platform and Zebrafish Platform) and international top-level expertise in vertebrate genome evolution. Ray-finned (Actinopterygii) fishes representing distant phylogenetic lineages are chosen: Teleostei, zebr afish (superorder: Ostariophysi), Atlantic cod (superorder: Paracanthopterygii), Atlantic halibut (superorder: Acanthopterygii), and spotted gar (Holostei), a living fossil, serving as an outgroup that diverged from the three other species before the tele ost genome duplication event. Comparative study performed on the four species will give reliable insight to evaluate principles that are universal or species specific for the investigated processes. We will focus on features that are so far poorly invest igated, such as occurrence of size variants in miRNAs and their differential patterning; the unknown role of miRNA 3-prime-end modifications; and the occurrence of very short ncRNA sequences in transcriptome datasets. Also, we will focus on germline-speci fic ncRNAs to elucidate their role in germline specification with an applicative outcome to control reproduction in aquaculture species. The proposed research addresses fundamental questions in biology and medicine related to regulatory mechanisms of biol ogical processes. The project will generate new knowledge on ncRNA abundance, distribution, evolutionary conservation and germline development. The comparative study implies potential impact on farm fish species.