Identification of MicroRNAs from <em>Eugenia uniflora</em> by High-Throughput Sequencing and Bioinformatics Analysis

<div><h3>Background</h3><p>microRNAs or miRNAs are small non-coding regulatory RNAs that play important functions in the regulation of gene expression at the post-transcriptional level by targeting mRNAs for degradation or inhibiting protein translation. <em>Eugenia uniflora</em> is a plant native to tropical America with pharmacological and ecological importance, and there have been no previous studies concerning its gene expression and regulation. To date, no miRNAs have been reported in Myrtaceae species.</p> <h3>Results</h3><p>Small RNA and RNA-seq libraries were constructed to identify miRNAs and pre-miRNAs in <em>Eugenia uniflora</em>. Solexa technology was used to perform high throughput sequencing of the library, and the data obtained were analyzed using bioinformatics tools. From 14,489,131 small RNA clean reads, we obtained 1,852,722 mature miRNA sequences representing 45 conserved families that have been identified in other plant species. Further analysis using contigs assembled from RNA-seq allowed the prediction of secondary structures of 25 known and 17 novel pre-miRNAs. The expression of twenty-seven identified miRNAs was also validated using RT-PCR assays. Potential targets were predicted for the most abundant mature miRNAs in the identified pre-miRNAs based on sequence homology.</p> <h3>Conclusions</h3><p>This study is the first large scale identification of miRNAs and their potential targets from a species of the Myrtaceae family without genomic sequence resources. Our study provides more information about the evolutionary conservation of the regulatory network of miRNAs in plants and highlights species-specific miRNAs.</p> </div

Number of identified miRNAs in each conserved miRNA family in plants.

<p>The values above the bars indicate the number of members identified in each conserved miRNA family.</p

Predicted targets of novel miRNAs in <i>E. uniflora.</i>

*<p>psRNATarget value.</p

New putative miRNA precursors identified in <i>E. uniflora</i>.

<p>MFE: minimal folding free energy (kcal/mol); AMFE: Adjusted MFE; MFEI: minimal folding free energy index; ND: no detected.</p

Predicted secondary structures of conserved and novel miRNAs of <i>E. uniflora</i>.

<p>Secondary structures of the precursors eun-MIR535-1 and eun-nMIR012, their locations and the expression of small RNAs mapped onto these precursors. Sequences of the most abundant mature miRNAs in the 5p and 3p arms are labeled in purple and red, respectively. Values on the left side of the miRNA sequence represent read counts in the leaf library.</p

Pre-miRNAs identified in <i>E. uniflora</i> with sequence similarities to plant conserved miRNA families.

<p>MFE: minimal folding free energy (kcal/mol); AMFE: Adjusted MFE; MFEI: minimal folding free energy index; ND: no detected.</p

Summary of data from sequencing of <i>E. uniflora</i> small RNA library.

*<p>Reads with high quality with lenghts of 1 to 44 nt.</p

Length distribution and diversity of small RNA reads in the <i>E. uniflora</i> leaf library.

<p>Length distribution and diversity of small RNA reads in the <i>E. uniflora</i> leaf library.</p