Abstract
Micro-RNAs (miRNAs) are now recognized as a major class of developmental regulators. Sequences of many miRNAs are highly conserved, yet they often exhibit temporal and spatial heterogeneity in expression among species and have been proposed as an important reservoir for adaptive evolution and divergence. With this in mind we studied miRNA expression during embryonic development of offspring from two contrasting morphs of the highly polymorphic salmonid Arctic charr (Salvelinus alpinus), a small benthic morph from Lake Thingvallavatn (SB) and an aquaculture stock (AC). These morphs differ extensively in morphology and adult body size. We established offspring groups of the two morphs and sampled at several time points during development. Four time points (3 embryonic and one just before first feeding) were selected for high-throughput small-RNA sequencing. We identified a total of 326 conserved and 427 novel miRNA candidates in Arctic charr, of which 51 conserved and 6 novel miRNA candidates were differentially expressed among developmental stages. Furthermore, 53 known and 19 novel miRNAs showed significantly different levels of expression in the two contrasting morphs. Hierarchical clustering of the 53 conserved miRNAs revealed that the expression differences are confined to the embryonic stages, where miRNAs such as sal-miR-130, 30, 451, 133, 26 and 199a were highly expressed in AC, whereas sal-miR-146, 183, 206 and 196a were highly expressed in SB embryos. The majority of these miRNAs have previously been found to be involved in key developmental processes in other species such as development of brain and sensory epithelia, skeletogenesis and myogenesis. Four of the novel miRNA candidates were only detected in either AC or SB. miRNA candidates identified in this study will be combined with available mRNA expression data to identify potential targets and involvement in developmental regulation.
Highlights
Since the initial discoveries of lin-4 and let-7 miRNAs have emerged as key regulators of animal development
Small RNA sequencing descriptive statistics In order to identify miRNAs involved in Arctic charr development and morph differences, we made 8 small RNA libraries from four developmental time-points of two contrasting morphs of Arctic charr
Annotation of the collapsed reads using the Rfam database confirmed that our small RNA libraries were highly enriched with miRNAs (Table 2)
Summary
Since the initial discoveries of lin-4 and let-7 miRNAs have emerged as key regulators of animal development (reviewed in [1,2]). These small (,22 nt) non coding RNAs, regulate gene expression by inducing mRNA degradation or translational repression, making for a specific and ‘‘fine-tunable’’ response (reviewed in [3]). A common feature of all miRNA genes, regardless of their genome location, is the folding of their primary transcript into a stem-loop structure. This hairpin structure is recognized and converted into a miRNAmiRNA* duplex by the miRNA processing machinery (see [5]). In most cases of miRNA mediated gene regulation the target repertoire is determined by the ‘‘seed’’ region (nt 2-8 located at the 5’ end of the mature miRNA) of the miRNA [7]
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