Abstract
BackgroundAmong the hundreds of genes encoding miRNAs in plants reported, much more were predicted by numerous computational methods. However, unlike protein-coding genes defined by start and stop codons, the ends of miRNA molecules do not have characteristics that can be used to define the mature miRNAs exactly, which made computational miRNA prediction methods often cannot predict the accurate location of the mature miRNA in a precursor with nucleotide-level precision. To our knowledge, there haven't been reports about comprehensive strategies determining the precise sequences, especially two termini, of these miRNAs.MethodsIn this study, we report an efficient method to determine the precise sequences of computationally predicted microRNAs (miRNAs) that combines miRNA-enriched library preparation, two specific 5′ and 3′ miRNA RACE (miR-RACE) PCR reactions, and sequence-directed cloning, in which the most challenging step is the two specific gene specific primers designed for the two RACE reactions. miRNA-mediated mRNA cleavage by RLM-5′ RACE and sequencing were carried out to validate the miRNAs detected. Real-time PCR was used to analyze the expression of each miRNA.ResultsThe efficiency of this newly developed method was validated using nine trifoliate orange (Poncirus trifoliata) miRNAs predicted computationally. The miRNAs computationally identified were validated by miR-RACE and sequencing. Quantitative analysis showed that they have variable expression. Eight target genes have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in Poncirus trifoliate.ConclusionThe efficient and powerful approach developed herein can be successfully used to validate the sequences of miRNAs, especially the termini, which depict the complete miRNA sequence in the computationally predicted precursor.
Highlights
One of the most important developments in molecular biology over the past two decades is the emerging picture of a new layer of gene regulation under the control of small yet versatile RNAs [1]
The first parameter was that the primers covered 17 nucleotides of the candidate mature miRNAs, and these 17 nucleotides were specific to the corresponding miRNA and met the criterion for the minimum number of nucleotides of a regular PCR primer
The second parameter was that mismatches between the sequence of the specific primer and that of the end sequence of the real miRNAs were allowed, and that these mismatches should not influence the PCR amplification, similar to the principle employed in site-directed mutagenesis [15,16] and in the addition of restriction sites to the termini of amplified DNA employed in recombinant DNA technology [17]
Summary
One of the most important developments in molecular biology over the past two decades is the emerging picture of a new layer of gene regulation under the control of small yet versatile RNAs [1]. MicroRNAs (miRNAs) are produced from partially complementary dsRNA precursor molecules [3,4]. These plant miRNAs are the best-characterized sRNAs, and the pathways by which they are generated and their roles in gene regulation have been well documented [2,3,5]. Among the hundreds of genes encoding miRNAs in plants reported, much more were predicted by numerous computational methods. Unlike protein-coding genes defined by start and stop codons, the ends of miRNA molecules do not have characteristics that can be used to define the mature miRNAs exactly, which made computational miRNA prediction methods often cannot predict the accurate location of the mature miRNA in a precursor with nucleotidelevel precision. There haven’t been reports about comprehensive strategies determining the precise sequences, especially two termini, of these miRNAs
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