Computational identification and characterization of microRNAs and their targets in Penaeus monodon

Study on shrimp miRNAs was limited and just 7 mature miRNA sequences of Marsupenaeus japonicus are deposited in mirBase database. In this study, miRNAs and their target gene candidates were computationally identified from shrimp Penaeu s monodon and then experimentally validated. Using 39 908 expressed sequence tags (ESTs) and 21 124 genome survey sequences (GSSs) of P. monodon (pmo) as reference dataset, a comprehensive approach based on inter-species homolog search was employed to investigate the candidate miRNAs (i.e. pmo-miRNA). A total of eight miRNAs belonging to 7 families were computationally identified and five out of them were subsequently validated by PCR and sequencing. Of these, pmo-miR-4961a, pmo-miR-4961b, pmo-miR-4979 and pmo-miR-3819 were first identified from shrimps. Both the mature pmo-miRNAs and the corresponding precursors were conserved among different species. Based on perfect or near-perfect match to the target region, the target gene candidates of pmomiRNAs were predicted from 10 331 mRNA sequences of P. monodon. A total of 20 genes were predicted as the targets of pmo-miR-4961a, pmo-miR-4961b, pmo-miR-4979 and pmo-miR-6492. Experimental validation by dual luciferase reporter assay confirmed the targeting between 3 pmo-miRNAs and one or two of their target genes, especially the pmo-miR-4979 which could significantly down-regulate the expression of target gene (JR226772). This study updates the miRNAs and their targets in P. monodon and lays a solid foundation for future RNAi study.