Discovery of Novel MicroRNAs in Rat Kidney Using Next Generation Sequencing and Microarray Validation

Fanxue Meng,Michael Hackenberg,Tao Chen,Jian Yan,Zhiguang Li,Neil R Smalheiser

doi:10.1371/journal.pone.0034394

Fanxue Meng, Michael Hackenberg + Show 4 more

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https://doi.org/10.1371/journal.pone.0034394

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Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that regulate a variety of biological processes. The latest version of the miRBase database (Release 18) includes 1,157 mouse and 680 rat mature miRNAs. Only one new rat mature miRNA was added to the rat miRNA database from version 16 to version 18 of miRBase, suggesting that many rat miRNAs remain to be discovered. Given the importance of rat as a model organism, discovery of the completed set of rat miRNAs is necessary for understanding rat miRNA regulation. In this study, next generation sequencing (NGS), microarray analysis and bioinformatics technologies were applied to discover novel miRNAs in rat kidneys. MiRanalyzer was utilized to analyze the sequences of the small RNAs generated from NGS analysis of rat kidney samples. Hundreds of novel miRNA candidates were examined according to the mappings of their reads to the rat genome, presence of sequences that can form a miRNA hairpin structure around the mapped locations, Dicer cleavage patterns, and the levels of their expression determined by both NGS and microarray analyses. Nine novel rat hairpin precursor miRNAs (pre-miRNA) were discovered with high confidence. Five of the novel pre-miRNAs are also reported in other species while four of them are rat specific. In summary, 9 novel pre-miRNAs (14 novel mature miRNAs) were identified via combination of NGS, microarray and bioinformatics high-throughput technologies.

Highlights

MicroRNAs are small non-coding RNAs of,22 nucleotides in length and ubiquitously present in plant and animal cells [1]. miRNAs play an important role in the post-transcriptional regulation of gene expression via binding to the 39 UTR region of the target mRNAs, resulting in mRNA degradation or translation inhibition [2]
Recognition of Rat Homologous Novel miRNAs Small RNA transcriptomes of kidney samples from 8 rats, 4 treated with aristolochic acid (AA) and 4 untreated as control, were analyzed using next generation sequencing (NGS) (NGS data are available through Gene Expression Omnibus series accession numbers GSE33703)
Using samples from different animals as well as AA-treated and untreated rats should strengthen the discovery of novel miRNAs as accidental discovery due to fluctuations can be virtually discarded

Summary

Introduction

MicroRNAs (miRNAs) are small non-coding RNAs of ,22 nucleotides in length and ubiquitously present in plant and animal cells [1]. miRNAs play an important role in the post-transcriptional regulation of gene expression via binding to the 39 UTR region of the target mRNAs, resulting in mRNA degradation or translation inhibition [2]. Pri-miRNAs are processed by RNase Drosha to release approximately 70 nucleotides long miRNA precursors (pre-miRNAs) that have characteristic hairpin structures. RNase Dicer cleaves the premiRNA hairpin to generate a double-stranded miRNA duplex with a characteristic 39 2-nucleotide overhang. Mature variants generated from the same miRNA precursor contain different sequences from the mature and/or mature* sequence. These mature variants are named as isomirs [10]. The characteristic structures of these different stages of miRNA biogenesis, such as hairpin structures and mature* sequences, have been utilized for identification of novel miRNAs based on certain guidelines [11,12]. Due to the phylogenetic conservation of miRNAs, the requirements for defining homologous miRNAs are generally less strict than those for speciesspecific miRNAs such as those found in rats only [11,12]

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