Functional analysis of microRNA activity in Brugia malayi

• Insertion of a recognition site for a microRNA (miRNA) reduces reporter gene expression in Brugia malayi.
• Mutation of the seed and 5′ end sequences of the miRNA recognition site partially restore expression.
• miRNA target prediction algorithms developed for other organisms can predict miRNA targets in B. malayi.
• Transient transfection of B. malayi embryos can be used to functionally assay miRNA recognition sites.

The complement of the Brugia malayi microRNA-71 was inserted into the 3′ untranslated region of a reporter plasmid, resulting in a decrease in reporter activity. Mutation of the seed sequence restored activity. Insertion of the 3′ untranslated regions from two algorithm-predicted putative target genes into the reporter resulted in a similar decrease in activity; mutation of the predicted target sequences restored activity. These experiments demonstrate that B. malayi microRNA targets may be predicted using current algorithms and describe a functional assay to confirm predicted targets.