Genetics and Breeding Research Unit, United States Department of Agriculture/Agricultural Research Service/USDA/Meat Animal Research Center, Nebraska, United States of America.
PLoS One. 2012;7(7):e42039. doi: 10.1371/journal.pone.0042039. Epub 2012 Jul 27.
MicroRNA are a class of small RNAs that regulate gene expression by inhibiting translation of protein encoding transcripts through targeting of a microRNA-protein complex by base-pairing of the microRNA sequence to cognate recognition sequences in the 3' untranslated region (UTR) of the mRNA. Target identification for a given microRNA sequence is generally accomplished by informatics analysis of predicted mRNA sequences present in the genome or in databases of transcript sequence for the tissue of interest. However, gene models for porcine skeletal muscle transcripts in current databases, specifically complete sequence of the 3' UTR, are inadequate for this exercise.
METHODOLOGY/PRINCIPAL FINDINGS: To provide data necessary to identify gene targets for microRNA in porcine skeletal muscle, normalized cDNA libraries were sequenced using Roche 454 GS-FLX pyrosequencing and de novo assembly of transcripts enriched in the 3' UTR was performed using the MIRA sequence assembly program. Over 725 million bases of sequence were generated, which assembled into 18,202 contigs. Sequence reads were mapped to a 3' UTR database containing porcine sequences. The 3' UTR that mapped to the database were examined to predict targets for previously identified microRNA that had been separately sequenced from the same porcine muscle sample used to generate the cDNA libraries. For genes with microRNA-targeted 3' UTR, KEGG pathways were computationally determined in order to identify potential functional effects of these microRNA-targeted transcripts.
Through next-generation sequencing of transcripts expressed in skeletal muscle, mapping reads to a 3' UTR database, and prediction of microRNA target sites in the 3' UTR, our results identified genes expressed in porcine skeletal muscle and predicted the microRNA that target these genes. Additionally, identification of pathways regulated by these microRNA-targeted genes provides us with a set of genes that can be further evaluated for their potential role in skeletal muscle development and growth.
miRNA 是一类通过 miRNA 序列与 mRNA 3'UTR 中的互补识别序列碱基配对，形成 miRNA-蛋白复合物，从而抑制蛋白编码转录物翻译的小 RNA。给定 miRNA 序列的靶标识别通常通过对基因组中或感兴趣组织的转录物序列数据库中存在的预测 mRNA 序列进行计算分析来完成。然而，目前数据库中的猪骨骼肌转录物基因模型，特别是 3'UTR 的完整序列，不足以进行这项研究。
方法/主要发现：为了提供鉴定猪骨骼肌中 miRNA 基因靶标的必要数据，我们使用 Roche 454 GS-FLX 焦磷酸测序对正常化的 cDNA 文库进行测序，并使用 MIRA 序列组装程序对富含 3'UTR 的转录物进行从头组装。生成了超过 7.25 亿个碱基对序列，组装成 18202 个 contigs。将序列读段映射到包含猪序列的 3'UTR 数据库中。将映射到数据库的 3'UTR 进行检查，以预测之前从用于生成 cDNA 文库的相同猪肌肉样本中单独测序的已鉴定 miRNA 的靶标。对于具有 miRNA 靶向 3'UTR 的基因，通过计算确定 KEGG 途径，以确定这些 miRNA 靶向转录物的潜在功能影响。
通过骨骼肌中表达的转录物的下一代测序、将读段映射到 3'UTR 数据库以及预测 3'UTR 中的 miRNA 靶标位点，我们的结果鉴定了在猪骨骼肌中表达的基因，并预测了靶向这些基因的 miRNA。此外，鉴定受这些 miRNA 靶向基因调控的途径为我们提供了一组可以进一步评估其在骨骼肌发育和生长中潜在作用的基因。
