Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158-2280, USA.
Cell Microbiol. 2012 Sep;14(9):1455-73. doi: 10.1111/j.1462-5822.2012.01811.x. Epub 2012 May 23.
In the current investigation, we analysed all the known small nucleolar RNAs (snoRNAs) in the deeply branching protozoan parasite Giardia lamblia for potential microRNAs (miRNAs) that might be derived from them. Two putative miRNAs have since been identified by Northern blot, primer extension, 3' RACE and co-immunoprecipitation with Giardia Argonaute (GlAgo), and designated miR6 and miR10. Giardia Dicer (GlDcr) is capable of processing the snoRNAs into the corresponding miRNAs in vitro. Potential miR6 and miR10 binding sites in Giardia genome were predicted bio-informatically. A miR6 binding site was found at the 3' untranslated regions (UTR) of 44 variant surface protein (vsp) genes, whereas a miR10 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 miR6 and miR10. A reporter mRNA tagged with the 3' end of vsp1267, which contains the target sites for both miRNAs, was translationally repressed by both miRNAs in Giardia. Episomal expression of an N-terminal c-myc tagged VSP1267 was found significantly repressed by introducing either miR6 or miR10 into the cells and the repressive effects were additive. When the 2'-O-methyl antisense oligos (ASOs) of either miR6 or miR10 was introduced, however, there was an enhancement of tagged VSP1267 expression suggesting an inhibition of the repressive effects of endogenous miR6 or miR10 by the ASOs. Of the total 220 vsp genes in Giardia, 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 Giardia.
在当前的研究中，我们分析了深度分支原生动物寄生虫贾第虫中所有已知的小核仁 RNA（snoRNA），以寻找可能源自它们的微小 RNA（miRNA）。此后，通过 Northern blot、引物延伸、3' RACE 和与贾第虫 Argonaute（GlAgo）的共免疫沉淀，鉴定了两个假定的 miRNA，并分别命名为 miR6 和 miR10。贾第虫 Dicer（GlDcr）能够在体外将 snoRNA 加工成相应的 miRNA。通过生物信息学预测了贾第虫基因组中潜在的 miR6 和 miR10 结合位点。在 44 个变异表面蛋白（vsp）基因的 3'非翻译区（UTR）发现了 miR6 结合位点，而在 159 个 vsp 开放阅读框的 3'端发现了 miR10 结合位点。其中 33 个 vsp 基因包含 miR6 和 miR10 的结合位点。标记有 vsp1267 3'端的报告 mRNA，该基因包含两个 miRNA 的靶位，在贾第虫中，这两个 miRNA 都能抑制其翻译。在细胞中引入 miR6 或 miR10 时，带有 N 端 c-myc 标记的 VSP1267 的外显子表达显著受到抑制，并且这种抑制作用是累加的。然而，当引入 miR6 或 miR10 的 2'-O-甲基反义寡核苷酸（ASO）时，标记的 VSP1267 表达增强，这表明 ASO 抑制了内源性 miR6 或 miR10 的抑制作用。在贾第虫的总共 220 个 vsp 基因中，我们现在发现 178 个携带了目前已鉴定出的所有 miRNA 的假定结合位点，这表明 miRNA 可能是贾第虫 VSP 表达的调节剂。
