Differential expression patterns of conserved miRNAs and isomiRs during Atlantic halibut development

Abstract

Background: MicroRNAs (miRNAs) play a major role in animal ontogenesis. Size variants of miRNAs, isomiRs, are observed along with the main miRNA types, but their origin and possible biological role are uncovered yet. Developmental profiles of miRNAs have been reported in few fish species only and, to our knowledge, differential expressions of isomiRs have not yet been shown during fish development. Atlantic halibut, Hippoglossus hippoglossus L., undergoes dramatic metamorphosis during early development from symmetrical pelagic larval stage to unsymmetrical flatfish. No data exist on role of miRNAs in halibut metamorphosis.

Results: miRNA profiling using SOLiD deep sequencing technology revealed a total of 199 conserved, one novel antisense, and one miRNA* mature form. Digital expression profiles of selected miRNAs were validated using reverse transcription quantitative PCR. We found developmental transition-specific miRNA expression. Expression of some miRNA* exceeded the guide strand miRNA. We revealed that nucleotide truncations and/or additions at the 3' end of mature miRNAs resulted in size variants showing differential expression patterns during the development in a number of miRNA families. We confirmed the presence of isomiRs by cloning and Sanger sequencing. Also, we found inverse relationship between expression levels of sense/antisense miRNAs during halibut development.

Conclusion: Developmental transitions during early development of Atlantic halibut are associated with expression of certain miRNA types. IsomiRs are abundant and often show differential expression during the development.

Figure 1

Read statistics for deep sequencing of Atlantic halibut small non-coding RNA. A) Quality score of reads averaged at each position for all samples; the box indicates inter-quartile range, where the lower boundary shows 1^st quartile and the top boundary shows the 3^rd quartile with the median line in between. B) Read length distribution after removal of adaptor sequences. The three peaks of read size frequencies represent very small RNA, miRNA/siRNA, and piRNA (see details in Results). C) Proportion of reads mapped to different databases excluding unmapped reads; other non-coding RNAs are < 1% in all stages. BL, EP, SS, HA, FF, EM, CM, and JU stand for blastula, epiboly, somitogenesis, hatching, first feeding, early metamorphosis, climax metamorphosis and juvenile developmental stages, respectively. D) Overall average mismatches, missed and added bases in halibut mature miRNAs compared to mature miRBase reference sequences.

Figure 2

The heatmap of identified miRNAs during Atlantic halibut early ontogenesis (see Figure 1C legend for abbreviations). The heatmap is obtained using R program version 2.12.0 (Euclidean distance, average linkage algorithm). There are 199 rows and 8 columns corresponding to each miRNA and developmental stage, respectively. The heatmap was drawn on log 2 normalized read counts in relation to the expression at hatching stage as a reference (no color), and downregulation and upregulation were denoted with shades of green and red, respectively.

Figure 3

Normalized read counts of selected miRNAs during eight stages of Atlantic halibut development. See Figure 1C legend for specification of developmental stages.

Figure 4

Relative expression of selected miRNAs using RT-qPCR. Different letters indicate statistically significant differences (P < 0.05) between means. Error bar is S.E.

Figure 5

Relative counts per million for guide and passenger strand miRNAs. See Figure 1C legend for developmental stages label descriptions.

Figure 6

IsomiRs expression pattern for selected miRNAs through early developmental stages of Atlantic halibut. The reference sequences are in bold italic. Y-axis is a relative frequency. Developmental stages are specified in Figure 1C legend

Figure 7

A) Atlantic halibut mir-147 precursor sequence in comparison with human, rat, chicken, and cattle precursor sequences. B) Alignment and sequence of novel antisense miRNA in Atlantic halibut. Mature guiding strand miRNA sequences are indicated in italic and bold, while miRNA* sequences are underlined. Differences in nucleotide among species are highlighted gray. Star (*) indicates the conservation among all species, whereas dot (.) indicates partial conservation. Numbers on the right indicate overall SOLiD read counts for the respective sequence.