Abstract
During early animal ontogenesis, a plethora of small non-coding RNAs (sncRNAs) are greatly expressed and have been shown to be involved in several regulatory pathways vital to proper development. The rapid advancements in sequencing and computing methodologies in the last decade have paved the way for the production of sequencing data in a broad range of organisms, including teleost species. Consequently, this has led to the discovery of sncRNAs as well as the potentially novel roles of sncRNA in gene regulation. Among the several classes of sncRNAs, microRNAs (miRNAs) have, in particular, been shown to play a key role in development. The present work aims to identify the miRNAs that play important roles during early European sea bass (Dicentrarchus labrax) development. The European sea bass is a species of high commercial impact in European and especially Mediterranean aquaculture. This study reports, for the first time, the identification and characterization of small RNAs that play a part in the 10 developmental stages (from morula to all fins) of the European sea bass. From 10 developmental stages, more than 135 million reads, generated by next-generation sequencing, were retrieved from publicly available databases as well as newly generated. The analysis resulted in about 2,000 sample grouped reads, and their subsequently annotation revealed that the majority of transcripts belonged to the class of miRNAs followed by small nuclear RNAs and small nucleolar RNAs. The analysis of small RNA expression among the developmental stages under study revealed that miRNAs are active throughout development, with the main activity occurring after the earlier stages (morula and 50% epiboly) and at the later stages (first feeding, flexion, and all fins). Furthermore, investigating miRNAs exclusively expressed in one of the stages unraveled five miRNAs with a higher abundance only in the morula stage (miR-155, miR-430a, d1, d2, and miR-458), indicating possible important key roles of those miRNAs in further embryonic development. An additional target search showed putative miRNA-mRNA interactions with possible direct and indirect regulatory functions of the identified miRNAs.
Highlights
Non-coding RNAs are involved in several different regulatory pathways
All experiments/methods in the present study were performed in accordance with the approved guidelines and regulations from the Hellenic Center for Marine Research (HCMR) Institutional Animal Care and Use Committee following the three Rs (Replacement, Reduction, and Refinement) guiding principles for more ethical use of animals in testing, which was first described by Russell and Burch in 1959 (EU Directive 2010/63)
Total RNA was extracted from all developmental stages using Nucleospin miRNA Kit for the isolation of small RNA and mRNA
Summary
Non-coding RNAs (ncRNAs) are involved in several different regulatory pathways. In the last decade, their importance has been demonstrated in a broad range of organisms, including teleost species (Herkenhoff et al, 2018). The rapid advancements in sequencing and computing methodologies have significantly enhanced the discovery of new miRNAs in humans and model species, such as the zebrafish (Danio rerio; Mishima, 2012), and in non-model species, such as the Atlantic halibut (Hippoglossus hippoglossus) (Bizuayehu et al, 2012a) and the catfish (Ictalurus punctatus) (Barozai, 2012). Besides their functional importance in diverse biological processes, it has been shown that miRNAs are essential for vertebrate development (Wienholds and Plasterk, 2005). The dynamics of miRNA expression during early ontogeny in nonmodel fish species, such as the Atlantic halibut (Bizuayehu et al, 2012b), turbot (Scophthalmus maximus); (Robledo et al, 2017), and Senegalese sole (Solea senegalensis; Campos et al, 2014), have been reported
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