The profile of snoRNA ‐derived microRNAs that regulate expression of variant surface proteins in G iardia lamblia

Summary In the current investigation, we analysed all the known small nucleolar RNAs ( snoRNAs ) in the deeply branching protozoan parasite G iardia lamblia for potential microRNAs ( miRNAs ) that might be derived from them. Two putative miRNAs have since been identified by N orthern blot, primer extension, 3′ RACE and co‐immunoprecipitation with G iardia A rgonaute ( GlAgo ), and designated miR 6 and miR 10. G iardia D icer ( GlDcr ) is capable of processing the snoRNAs into the corresponding miRNAs in vitro . Potential miR 6 and miR 10 binding sites in G iardia genome were predicted bio‐informatically. A miR 6 binding site was found at the 3′ untranslated regions ( UTR ) of 44 variant surface protein ( vsp ) genes, whereas a miR 10 binding site was identified at the 3′ end of 159 vsp open‐reading frames. Thirty‐three of these vsp genes turned out to contain binding sites for both miR 6 and miR 10. A reporter mRNA tagged with the 3′ end of vsp 1267, which contains the target sites for both miRNAs , was translationally repressed by both miRNAs in G iardia . Episomal expression of an N ‐terminal c‐myc tagged VSP 1267 was found significantly repressed by introducing either miR 6 or miR 10 into the cells and the repressive effects were additive. When the 2′‐ O ‐methyl antisense oligos ( ASOs ) of either miR 6 or miR 10 was introduced, however, there was an enhancement of tagged VSP 1267 expression suggesting an inhibition of the repressive effects of endogenous miR 6 or miR 10 by the ASOs . Of the total 220 vsp genes in G iardia , we have now found 178 of them carrying putative binding sites for all the miRNAs that have been currently identified, suggesting that miRNAs are likely the regulators of VSP expression in G iardia .