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

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

Short hairpin RNA-mediated knockdown of protein expression in Entamoeba histolytica

<p>Abstract</p> <p>Background</p> <p><it>Entamoeba histolytica </it>is an intestinal protozoan parasite of humans. The genome has been sequenced, but the study of individual gene products has been hampered by the lack of the ability to generate gene knockouts. We chose to test the use of RNA interference to knock down gene expression in <it>Entamoeba histolytica</it>.</p> <p>Results</p> <p>An episomal vector-based system, using the <it>E. histolytica </it>U6 promoter to drive expression of 29-basepair short hairpin RNAs, was developed to target protein-encoding genes in <it>E. histolytica</it>. The short hairpin RNAs successfully knocked down protein levels of all three unrelated genes tested with this system: Igl, the intermediate subunit of the galactose- and N-acetyl-D-galactosamine-inhibitable lectin; the transcription factor URE3-BP; and the membrane binding protein EhC2A. Igl levels were reduced by 72%, URE3-BP by 89%, and EhC2A by 97%.</p> <p>Conclusion</p> <p>Use of the U6 promoter to drive expression of 29-basepair short hairpin RNAs is effective at knocking down protein expression for unrelated genes in <it>Entamoeba histolytica</it>, providing a useful tool for the study of this parasite.</p

Mapped small RNAs in <i>E</i>. <i>invadens</i> show relatively greater occupancy of promoter regions compared to <i>E</i>. <i>histolytica</i>.

<p>Small RNA sequences from <i>E</i>. <i>histolytica</i> and <i>E</i>. <i>invadens</i> trophozoites were aligned to the region from -100 to +100 (relative to ATG) of each annotated ORF. The frequency of antisense RNA 5' ends (for genes with ≥20 antisense RNAs) at each position was plotted. The ratio of small RNAs in the upstream region (-100 to ATG) to small RNAs in the coding region (ATG to +100) of each ORF is shown.</p

Composition of small RNA libraries from <i>E</i>. <i>histolytica</i> under basal and stress conditions.

<p>Sequenced libraries from each condition were processed to remove barcodes, size selected for reads >15nt or <40nt, duplicate reads removed and the remaining unique reads sequentially mapped against each sequence type shown. Note that a small number of reads mapped both antisense and sense to transcripts, hence the total number of reads mapped to “transcript” is lower than the total number of reads mapping antisense plus sense. Total number of reads and percent of unique mapped reads are shown.</p

A custom-made antibody to <i>Entamoeba</i> AGO2-2 detects a 110kDa protein, which is enriched in the nucleus.

<p><b>(A)</b> Western Blot detects a band at 110 kDa using α-EhAGO2-2 antibody on <i>E</i>. <i>invadens</i> whole cell lysate, cytosolic fraction and nuclear fractions. <b>(B)</b> Immunofluorescence assay on <i>E</i>. <i>invadens</i> trophozoites showed nuclear enriched signal, which co-localizes with DAPI. Some cytoplasmic signal is also noted.</p

Mapping of <i>E</i>. <i>invadens</i> small RNAs to transposons and repetitive elements.

<p>Unique reads from <i>E</i>. <i>invadens</i> trophozoites, after removal of tRNA and rRNA mapped reads, were aligned to each type of transposable and repetitive element found in the <i>E</i>. <i>invadens</i> genome. Total read number, percent of unique reads, and percent of the <i>E</i>. <i>invadens</i> genome that each element represents (adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134481#pone.0134481.ref030" target="_blank">30</a>]) are shown.</p

High Throughput Sequencing of <i>Entamoeba</i> 27nt Small RNA Population Reveals Role in Permanent Gene Silencing But No Effect on Regulating Gene Expression Changes during Stage Conversion, Oxidative, or Heat Shock Stress

<div><p>The human parasite <i>Entamoeba histolytica</i> has an active RNA interference (RNAi) pathway with an extensive repertoire of 27nt small RNAs that silence genes. However the role of this pathway in regulating amebic biology remains unknown. In this study, we address whether silencing via 27nt small RNAs may be a mechanism for controlling gene expression changes during conversion between the trophozoite and cyst stages of the parasite. We sequenced small RNA libraries generated from trophozoites, early cysts, mature cysts, and excysting cells and mapped them to the <i>E</i>. <i>invadens</i> genome. Our results show that, as in <i>E</i>. <i>histolytica</i>, small RNAs in <i>E</i>. <i>invadens</i> are largely ~27nt in length, have an unusual 5'-polyphosphate structure and mediate gene silencing. However, when comparing the libraries from each developmental time-point we found few changes in the composition of the small RNA populations. Furthermore, genes targeted by small RNAs were permanently silenced with no changes in transcript abundance during development. Thus, the <i>E</i>. <i>invadens</i> 27nt small RNA population does not mediate gene expression changes during development. In order to assess the generalizability of our observations, we examined whether small RNAs may be regulating gene expression changes during stress response in <i>E</i>. <i>histolytica</i>. Comparison of the 27nt small RNA populations from <i>E</i>. <i>histolytica</i> trophozoites from basal conditions, or after heat shock or exposure to oxidative stress showed few differences. Similar to data in <i>E</i>. <i>invadens</i> development, genes targeted by small RNAs were consistently silenced and did not change expression under tested stress conditions. Thus, the biological roles of the 27nt small RNA population in <i>Entamoeba</i> remain elusive. However, as the first characterization of the RNAi pathway in <i>E</i>. <i>invadens</i> these data serve as a useful resource for the study of <i>Entamoeba</i> development and open the door to the development of RNAi-based gene silencing tools in <i>E</i>. <i>invadens</i>.</p></div

Comparison of genes with abundant small RNAs during <i>E</i>. <i>invadens</i> development.

<p><b>(A)</b> Venn diagram of genes with ≥20 AS small RNAs mapped at each time-point. Note that the majority of genes with small RNAs are represented in all time points. Due to the smaller number of reads in the trophozoite library, all genes with ≥20 small RNAs in that library are present in the other libraries; hence this time point was not included in the diagram. <b>(B)</b> Genes with ≥20 AS small RNAs are not expressed during development. Expression data are based on RNA-Seq from <i>E</i>. <i>invadens</i> trophozoites, early cysts (24h), mature cysts (72h) and excysting parasites are shown as Fragments per kilobase per million mapped reads (FPKM). The 789 genes to which ≥20 AS small RNAs mapped in any timepoint had very low expression under all timepoints (red), compared to expression of all genes (blue), indicating that they are not regulated during development.</p