Small RNAs with 5′-Polyphosphate Termini Associate with a Piwi-Related Protein and Regulate Gene Expression in the Single-Celled Eukaryote Entamoeba histolytica

Abstract

Small interfering RNAs regulate gene expression in diverse biological processes, including heterochromatin formation and DNA elimination, developmental regulation, and cell differentiation. In the single-celled eukaryote Entamoeba histolytica, we have identified a population of small RNAs of 27 nt size that (i) have 5′-polyphosphate termini, (ii) map antisense to genes, and (iii) associate with an E. histolytica Piwi-related protein. Whole genome microarray expression analysis revealed that essentially all genes to which antisense small RNAs map were not expressed under trophozoite conditions, the parasite stage from which the small RNAs were cloned. However, a number of these genes were expressed in other E. histolytica strains with an inverse correlation between small RNA and gene expression level, suggesting that these small RNAs mediate silencing of the cognate gene. Overall, our results demonstrate that E. histolytica has an abundant 27 nt small RNA population, with features similar to secondary siRNAs from C. elegans, and which appear to regulate gene expression. These data indicate that a silencing pathway mediated by 5′-polyphosphate siRNAs extends to single-celled eukaryotic organisms.

Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway

“…E. histolytica strains containing trigger silencing or Myc overexpression constructs were previously generated (34,35). Briefly, the 19-Trigger strains, 19T-ROM1, 19T-Ago2-2, and 19T-RdRP1, contain a silencing construct that has a short Trigger region fused to the full-length EhROM1, EhAgo2-2, or EhRdRP1, respectively.…”

“…Despite its position as a deep-branching eukaryote, E. histolytica has a distinct and complex endogenous RNAi pathway mediated by 27-nucleotide 5Ј-poly-P small RNAs that map antisense to genes and mediate TGS via a nuclear-localized Argonaute protein (33,34). We have recently developed a robust silencing tool in ameba by harnessing the endogenous RNAi pathway.…”

A microRNA derived from an apparent canonical biogenesis pathway regulates variant surface protein gene expression in Giardia lamblia

“…Most, if not all, of the small RNAs thus carry a 59 phosphate. The 39 ends of the HA-GlAgo coimmunoprecipitated small RNAs were examined by b-elimination (Zhang et al 2008). Denaturing PAGE analysis of the reaction product showed a decrease in the size of RNAs following b-elimination (Fig.…”

Comparative transcriptome analysis of small noncoding RNAs in different stages of Trypanosoma brucei

“…In contrast, 5 These authors contributed equally to this work. 6 siRNAs are ubiquitous and abundant in many protozoa (Robinson and Beverley 2003;Ullu et al 2005;Zhang et al 2008;Patrick et al 2009;Lye et al 2010;Tschudi et al 2012), suggesting that siRNAs, but not miRNAs, may be the key molecules in the RNA interference (RNAi) pathway in protozoan species.…”