Faculty of Biosciences and Aquaculture, University of Nordland, Bodø, Norway.
PLoS One. 2013 Apr 23;8(4):e61378. doi: 10.1371/journal.pone.0061378. Print 2013.
microRNAs (miRNAs) are implicated in regulation of many cellular processes. miRNAs are processed to their mature functional form in a step-wise manner by multiple proteins and cofactors in the nucleus and cytoplasm. Many miRNAs are conserved across vertebrates. Mature miRNAs have recently been characterized in Atlantic halibut (Hippoglossus hippoglossus L.). The aim of this study was to identify and characterize precursor miRNA (pre-miRNAs) and miRNA targets in this non-model flatfish. Discovery of miRNA precursor forms and targets in non-model organisms is difficult because of limited source information available. Therefore, we have developed a methodology to overcome this limitation.
Genomic DNA and small transcriptome of Atlantic halibut were sequenced using Roche 454 pyrosequencing and SOLiD next generation sequencing (NGS), respectively. Identified pre- miRNAs were further validated with reverse-transcription PCR. miRNA targets were identified using miRanda and RNAhybrid target prediction tools using sequences from public databases. Some of miRNA targets were also identified using RACE-PCR. miRNA binding sites were validated with luciferase assay using the RTS34st cell line.
We obtained more than 1.3 M and 92 M sequence reads from 454 genomic DNA sequencing and SOLiD small RNA sequencing, respectively. We identified 34 known and 9 novel pre-miRNAs. We predicted a number of miRNA target genes involved in various biological pathways. miR-24 binding to kisspeptin 1 receptor-2 (kiss1-r2) was confirmed using luciferase assay.
This study demonstrates that identification of conserved and novel pre-miRNAs in a non-model vertebrate lacking substantial genomic resources can be performed by combining different next generation sequencing technologies. Our results indicate a wide conservation of miRNA precursors and involvement of miRNA in multiple regulatory pathways, and provide resources for further research on miRNA in non-model animals.
microRNAs (miRNAs) 参与调控许多细胞过程。miRNAs 由核和细胞质中的多种蛋白质和辅助因子逐步加工为成熟的有功能形式。许多 miRNA 在脊椎动物中是保守的。最近在大西洋比目鱼（Hippoglossus hippoglossus L.）中对成熟 miRNAs 进行了描述。本研究旨在鉴定和描述这种非模式扁形鱼中的前体 miRNA (pre-miRNAs) 和 miRNA 靶标。由于可用的源信息有限，因此在非模式生物中发现 miRNA 前体形式和靶标是困难的。因此，我们开发了一种克服此限制的方法。
使用 Roche 454 焦磷酸测序和 SOLiD 下一代测序（NGS）分别对大西洋比目鱼的基因组 DNA 和小转录组进行测序。使用反转录 PCR 进一步验证鉴定的 pre-miRNAs。使用 miRanda 和 RNAhybrid 靶标预测工具，使用公共数据库中的序列，鉴定 miRNA 靶标。还使用 RACE-PCR 鉴定了一些 miRNA 靶标。使用 RTS34st 细胞系的荧光素酶测定法验证 miRNA 结合位点。
我们从 454 基因组 DNA 测序和 SOLiD 小 RNA 测序中分别获得了超过 130 万个和 9200 万个序列读数。我们鉴定了 34 个已知和 9 个新的 pre-miRNAs。我们预测了许多涉及各种生物途径的 miRNA 靶基因。使用荧光素酶测定法证实了 miR-24 与 kisspeptin 1 受体-2 (kiss1-r2) 的结合。
本研究表明，在缺乏大量基因组资源的非模式脊椎动物中，可以通过结合不同的下一代测序技术来鉴定保守和新的 pre-miRNAs。我们的结果表明 miRNA 前体广泛保守，并且 miRNA 参与多个调节途径，并为非模式动物中 miRNA 的进一步研究提供了资源。
