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.
Regulation of gene expression can occur via multiple conserved pathways. One such mechanism is mediated by RNA molecules of about 21–24 nucleotides (called small RNAs), which can affect rates of RNA degradation or protein production. These small RNA molecules regulate diverse biological processes in a broad range of systems. The vast majority of the published literature about these molecules is from multi-cellular organisms. We have made a number of novel observations with respect to small RNA size, structure, and function in Entamoeba histolytica, a single-celled parasite and an important human pathogen. Our work has identified that E. histolytica has an abundant population of 27 nucleotide small RNAs, which have an unusual structure, indicating that they are generated by a relatively atypical mechanism. A substantial portion of these small RNAs are antisense to target genes and appear to silence them. These data establish a new paradigm for how gene expression is regulated in this organism. Furthermore, the identification of small RNAs with these structural characteristics dramatically broadens the evolutionary spectrum in which this phenomenon has been identified and indicates significant diversity and complexity of small RNAs and their functions in single-celled eukaryotes.