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. 2016 Apr 12:17:283.
doi: 10.1186/s12864-016-2606-5.

Identification and characterization of novel and conserved microRNAs in several tissues of the Chinese rare minnow (Gobiocypris rarus) based on illumina deep sequencing technology

Xiangsheng Hong  1   2 , Jianhui Qin  2 , Rui Chen  1   3 , Lilai Yuan  1   3 , Jinmiao Zha  4   5 , Zijian Wang  3
Affiliations

Identification and characterization of novel and conserved microRNAs in several tissues of the Chinese rare minnow (Gobiocypris rarus) based on illumina deep sequencing technology

Xiangsheng Hong et al. BMC Genomics. .

Abstract

Background: MicroRNAs (miRNAs), which comprise a large family of endogenous small non-coding RNA molecules, play important roles in the regulation of gene expression in various biological processes. The Chinese rare minnow (Gobiocypris rarus) is a Chinese native fish species and is used extensively as an experimental fish in China; however, relevant biological data, especially miRNA transcriptome data, have not been well documented. To discover conserved and potential novel miRNAs in Chinese rare minnows, a pool of equal amounts of RNA obtained from 6 different adult rare minnow tissues (brain, eye, gill, liver, muscle and heart) was sequenced using illumina deep sequencing technology.

Results: In the present study, 26,930,553 raw reads, representing 2,118,439 unique high-quality reads, were obtained from the pooled small RNA library. Using bioinformatics analysis, 352 conserved and 112 novel Chinese rare minnow miRNAs were first discovered and characterized in this study. Moreover, we found extensive sequence variations (isomiRs) in rare minnow miRNAs, including internal miRNA isomiRs and terminal isomiRs at both the 5' and 3' ends and nucleotide variants. Six conserved and 4 novel miRNAs were selected and validated in 6 different adult rare minnow tissues using quantitative real-time PCR (qPCR). The results showed that miR-30a, miR-30b, and Novel-37 are ubiquitously expressed in a variety of tissues. miR-16a, miR-9, miR-125b, miR-34a, and Novel-69 were predominantly expressed in the brain. Novel-115 and Novel-7 were highly expressed in gills, but were relatively weakly expressed in other tissues. These results provided the expression patterns of miRNA genes in Chinese rare minnow. Finally, based on bioinformatics predictions, we mainly found that Novel-94 and Novel-1b-5p were simultaneously targeted to the 3'UTR of Dmrt1, which controls sex determination and/or sexual differentiation in a variety of metazoans at different sites. Novel-29b targeted the 3'UTR of Foxl2, which is involved in the maintenance of ovarian function and the transcriptional regulation of gonadal differentiation-related genes. Novel-62 and Novel-53 targeted the 3'UTR of ERbeta1 and ERbeta2 (which regulate the transcription of target genes), respectively.

Conclusions: Rare minnow is a widely used model for assessing the risk of environmental pollution in China. Identifying and characterizing rare minnow miRNA genes is necessary to discover the biological function of miRNAs and to screen for new molecule biomarkers to assess the risk of environmental pollution in the future.

Keywords: Deep sequencing; Gobiocypris rarus; IsomiRs; Target prediction; microRNA.

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Figures

Fig. 1
Fig. 1
Length distribution of small RNA sequences. Sequence length distribution of total reads based on abundance; the most abundant size class was 22 nt, followed by 23 nt and 25 nt
Fig. 2
Fig. 2
Schematic representation of microRNA isomiR species
Fig. 3
Fig. 3
miR-9 sequence heterogeneity. a Significant sequence variation was detected among mature miRNA reads. The figure shows a compilation of miR-9 variants; the extent of variation at any particular nucleotide is indicated by the size of the font by WebLogo [74]. b (Below) The mature 5p miRNA sequence is shown in green, and the 3p miRNA sequence is shown in red; the adjacent genomic sequence is shown in black. Single nucleotide substitutions are highlighted in gray. The number of unique reads that are aligned to the putative precursor is listed on the right side of the figure. c A graphic illustration of the hairpin-loop precursor is shown on the right of the figure. The dominant cleavage sites are indicated using black arrows. Large arrows show the most abundant isomer cleavage sites, whereas small arrows indicate less abundant isomiR cleavage sites. The most abundant mature miRNAs (5′p and 3′p) are shown by the regions shown in green and red
Fig. 4
Fig. 4
Tissue distribution of the miRNAs analyzed using qPCR. Tissue type is shown on the X axis. The relative miRNA expression level is shown on the Y axis. The results are presented as fold changes; rare minnow 5S rRNA is used as an endogenous control, and the fold change is expressed relative to the expression in brain. Values are shown as mean ± standard deviation (n = 3)
Fig. 5
Fig. 5
A schematic illustration of miRNA and its putative binding sites in the 3′UTR of selected genes. Novel miRNAs target 6 rare minnow genes using a 3′UTR binding site; these genes include dmrt1, Foxl2, AR, ERalpha, ERbeta1 and ERbeta2, according to predictions made using miRanda, TargetScan, RNA22 and PITA (a local version). The obtained predictions were identical among the four algorithms. The miRNA:mRNA sequence alignments, together with the calculated score and the minimum free energy of the duplex, are presented above each target binding site. In the alignment, “|” refers to perfect complementary between bases, “-” represents a gap, and “:” represents a G:U wobble pair
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