Small RNAs with 59-Polyphosphate Termini Associate with a Piwi-Related Protein and Regulate Gene Expression in the Single-Celled Eukaryote Entamoeba histolytica Hanbang Zhang 1 , Gretchen M. Ehrenkaufer 1 , Justine M. Pompey 2 , Jason A. Hackney 1¤ , Upinder Singh 1,2 * 1 Division of Infectious Diseases, Department of Internal Medicine, Stanford University School of Medicine, Stanford, California, United States of America, 2 Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America 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 59-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 59-polyphosphate siRNAs extends to single-celled eukaryotic organisms. Citation: Zhang H, Ehrenkaufer GM, Pompey JM, Hackney JA, Singh U (2008) Small RNAs with 59-Polyphosphate Termini Associate with a Piwi-Related Protein and Regulate Gene Expression in the Single-Celled Eukaryote Entamoeba histolytica. PLoS Pathog 4(11): e1000219. doi:10.1371/journal.ppat.1000219 Editor: Patricia J. Johnson, University of California Los Angeles, United States of America Received June 19, 2008; Accepted October 28, 2008; Published November 28, 2008 Copyright: ß 2008 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Grant support is acknowledged for US (NIH grants AI-053724 and grant P30 DK56339 to the Stanford University Digestive Disease Center), HZ (NIH grants AI-053724, AI-069382, and P30 DK56339), GME (NIH grant AI-068899 and a Stanford University McCormick Fellowship), JMP (NIH training grant T32- AI07328), and JAH (NIH training grant T32-AI07328). The funders had no roles in the design or conduct of the study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: usingh@stanford.edu ¤ Current address: Department of Bioinformatics, Genentech, South San Francisco, California, United States of America Introduction Small RNAs mediate post-transcriptional gene silencing in a multitude of organisms and in diverse biological processes [1,2,3,4,5]. Two proteins central to the small RNA mediated gene silencing pathways are Dicer, an RNaseIII enzyme, which generates small RNAs and Argonaute, which associates with the small RNAs and target genes to mediate gene silencing. Multiple classes of small RNAs have recently been described including small interfering RNAs (siRNAs), microRNAs (miRNAs), trans-acting siRNAs (tasiRNAs), tiny noncoding RNAs (tncRNAs), small scan RNA (scRNA), repeat-associated small interfering RNA (ra- siRNA), piwi-interacting RNA (piRNA), and secondary siRNAs [6,7,8,9]. Some organisms have multiple populations of small RNAs associated with different mechanisms of gene regulation. Notably siRNAs, miRNAs, tasiRNAs, tncRNA, and scnRNA are all products of Dicer cleavage. In contrast, rasiRNA, piRNA, and secondary siRNA appear to be formed independent of Dicer processing [6,7,8,10]. Primary siRNAs are produced from long double stranded RNAs and can be endogenously derived from repetitive genomic regions, transposon elements, or regions with active antisense transcripts. Primary siRNAs are generated by Dicer processing, which generates a 59-monophosphate (59-monoP) and 39-hydroxyl (39- OH) structure. Primary siRNAs are subsequently loaded into Argonaute to mediate gene silencing but can also serve as the ‘‘trigger’’ to initiate RNA-dependent RNA polymerase (RdRP) generation of secondary siRNAs. In plants, secondary siRNAs, although generated by RdRP, are eventually processed by Dicer and thus the majority have the classic 59-monoP termini [9]. In C. elegans, secondary siRNAs have a 59-polyphosphate (59-polyP) structure, a feature not identified in any other siRNAs to date. C. elegans secondary siRNAs largely map antisense to genes, are biased towards the 59 side of primary trigger RNAs, and amplify gene silencing by their association with CSR-1, an Argonaute protein [7,8,10,11,12]. Because of their 59-polyP structure, C. elegans secondary siRNAs are most efficiently cloned in a 59- phosphate independent manner [7,8]. Entamoeba histolytica, a single celled eukaryote, is an important human pathogen and a leading parasitic cause of death worldwide [13]. The parasite has two stages in its life cycle: an invasive trophozoite form, which causes disease and a dormant cyst form, which transmits disease [14]. The genome of E. histolytica is predicted to encode a number of genes conserved in the RNAi pathway including three genes with Piwi and PAZ domains (EHI_186850, EHI_125650, and EHI_177170), and two genes with RdRP domains (EHI_139420 and EHI_179800) [15]. However, no obvious homologue of Dicer was identified, although PLoS Pathogens | www.plospathogens.org 1 November 2008 | Volume 4 | Issue 11 | e1000219