Identification and characterisation of primary microRNAs from apple (Malus domestica cv. Royal Gala) expressed sequence tags

Microribonucleic acids (miRNAs) are small, non-coding RNAs that play important regulatory roles by down-regulating target transcripts in a sequence-specific manner. The miRBase Registry (Release 8.2) lists 732 miRNAs from flowering plant species, with the majority identified from Arabidopsis, rice and poplar where genome sequence is available. In the absence of genomic sequence and on the basis that sequences of many miRNAs are conserved amongst divergent plant species, we analysed approximately 120,000 Malus domestica cv. Royal Gala expressed sequence tags (ESTs) and identified ten distinct sequences that could be classified into seven conserved plant miRNA families (miR156, miR159, miR162, miR167, miR172, miR393 and miR398). Secondary structure predictions showed these sequences have the characteristic fold-back structures of precursor miRNAs, and northern analysis validated the presence of these miRNA families within Royal Gala tissues. A number of the miRNAs were expressed constitutively in all tissues tested (miR159, miR162 and miR172), while others showed more restricted patterns of expression, being expressed primarily in leaf (miR398), expressed in leaf and floral bud tissue but down-regulated during fruit development (miR156 and miR167) or expressed in fungal pathogen-infected leaf tissue (miR393). Potential targets for six of the miRNA families were identified from the EST dataset and completely sequenced complementary deoxyribonucleic acids. In general, these targets encode proteins shown to be the targets of corresponding miRNAs in other plant species. Demonstrating cleavage of a number of the putative target transcripts within the region of miRNA/messenger RNA complementarity provided further evidence of the functionality of the identified Royal Gala miRNAs.