Influenza A Virus (H3N8) in Dogs with Respiratory Disease, Florida

Genetic and antigenic characterization established the uniqueness of this virus circulating in dogs

Influenza A Virus (H3N8) in Dogs with Respiratory Disease, Florida

Genetic and antigenic characterization established the uniqueness of this virus circulating in dogs

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

RNAi pathway genes are resistant to small RNA mediated gene silencing in the protozoan parasite Entamoeba histolytica.

The RNA interference pathway in the protist Entamoeba histolytica plays important roles in permanent gene silencing as well as in the regulation of virulence determinants. Recently, a novel RNA interference (RNAi)-based silencing technique was developed in this parasite that uses a gene endogenously silenced by small RNAs as a "trigger" to induce silencing of other genes that are fused to it. Fusion to a trigger gene induces the production of gene-specific antisense small RNAs, resulting in robust and permanent silencing of the cognate gene. This approach has silenced multiple genes including those involved in virulence and transcriptional regulation. We now demonstrate that all tested genes of the amebic RNAi pathway are unable to be silenced using the trigger approach, including Argonaute genes (Ago2-1, Ago2-2, and Ago2-3), RNaseIII, and RNA-dependent RNA polymerase (RdRP). In all situations (except for RdRP), fusion to a trigger successfully induces production of gene-specific antisense small RNAs to the cognate gene. These small RNAs are capable of silencing a target gene in trans, indicating that they are functional; despite this, however, they cannot silence the RNAi pathway genes. Interestingly, when a trigger is fused to RdRP, small RNA induction to RdRP does not occur, a unique phenotype hinting that either RdRP is highly resistant to being a target of small RNAs or that small RNA generation may be controlled by RdRP. The inability of the small RNA pathway to silence RNAi genes in E. histolytica, despite the generation of functional small RNAs to these loci suggest that epigenetic factors may protect certain genomic loci and thus determine susceptibility to small RNA mediated silencing

A Single RNaseIII Domain Protein from <i>Entamoeba histolytica</i> Has dsRNA Cleavage Activity and Can Help Mediate RNAi Gene Silencing in a Heterologous System

<div><p>Dicer enzymes process double-stranded RNA (dsRNA) into small RNAs that target gene silencing through the RNA interference (RNAi) pathway. Dicer enzymes are complex, multi-domain RNaseIII proteins, however structural minimalism of this protein has recently emerged in parasitic and fungal systems. The most minimal Dicer, <i>Saccharomyces castellii</i> Dicer1, has a single RNaseIII domain and two double stranded RNA binding domains. In the protozoan parasite <i>Entamoeba histolytica</i> 27nt small RNAs are abundant and mediate silencing, yet no canonical Dicer enzyme has been identified. Although EhRNaseIII does not exhibit robust dsRNA cleavage <i>in vitro</i>, it can process dsRNA in the RNAi-negative background of <i>Saccharomyces cerevisiae</i>, and in conjunction with <i>S</i>. <i>castellii</i> Argonaute1 can partially reconstitute the RNAi pathway. Thus, although EhRNaseIII lacks the domain architecture of canonical or minimal Dicer enzymes, it has dsRNA processing activity that contributes to gene silencing via RNAi. Our data advance the understanding of small RNA biogenesis in <i>Entamoeba</i> as well as broaden the spectrum of non-canonical Dicer enzymes that contribute to the RNAi pathway.</p></div

The fusion of the trigger to EhRdRP1 does not result in generation of AS sRNAs to EhRdRP1.

<p>High resolution Northern blot of trophozoites transfected with the trigger-EhRdRP1 plasmid. The lines A, B, and C, are three independently transfected cell lines maintained at 24 µg/ml G418. No RdRP1 AS sRNAs were detected in 75 µg of small-RNA enriched material from any of the three transfectant cell lines. EHI_118130 and EHI_197520 serve as controls.</p

Antisense sRNAs are detected to putative RNAi genes but do not silence the target genes.

<p>(<b>A</b>) High resolution Northern blot of parasites transfected with the trigger-EhAgo2-1 or the trigger-EhAgo2-3 plasmid. Cell lines maintained at 24 µg/ml G418. Gene-specific AS sRNAs to Ago2-1 or Ago2-3 are detected in the respective cell lines. AS sRNAs to EHI_188130 (loading control) and EHI_197520 (trigger) serve as controls. (<b>B</b>) High resolution Northern blot analysis of trigger-EhRNaseIII transfectants maintained in 6 µg/ml G418 or removed from drug selection for 1, 4, or 20 weeks. Abundant EhRNaseIII AS sRNAs are detected at all time points excluding the 20-week removal from drug sample. The 20-week sample shows a very faint population of EhRNaseIII AS sRNAs. Controls are the same as in (A). (<b>C</b>) Semi-quantitative RT-PCR of trigger-EhAgo2-1 transfectants at 24 µg/ml G418 shows an increase in EhAgo2-1 transcript abundance compared to untransfected cells. EHI_199600 was used as a loading control and -RT samples as negative controls. (<b>D</b>) Semi-quantitative RT-PCR shows an upregulation of EhAgo2-3 transcript in the trigger-EhAgo2-3 cell line at 24 µg/ml G418 compared to untransfected cells. Controls are the same as in (C). (<b>E</b>) Semi-quantitative RT-PCR of trigger-EhRNaseIII transfectants at 6 µg/ml G418 shows that the abundance of EhRNaseIII transcript is unaffected by the presence of gene-specific AS sRNAs. Controls are the same as in (C).</p

Schematic of model.

<p>(<b>A</b>) Functional AS sRNAs are generated to some amebic genes when fused to the trigger and the episomal and endogenous genes are silenced. sRNAs and silencing persist after plasmid removal and silencing can be extended to a fusion construct containing a portion of the endogenous gene. (Modified from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106477#pone.0106477-Morf2" target="_blank">[29]</a>). (<b>B</b>) Functional AS sRNAs to some amebic RNAi genes are induced when fused to the trigger but the endogenous gene is not silenced. Amebic RNAi sRNAs are not maintained after plasmid removal. (<b>C</b>) No AS sRNAs are made to EhRdRP1, and therefore EhRdRP1 is not silenced.</p

Schematic of RNAi reconstitution assays in <i>S</i>. <i>cerevisiae</i>.

<p>Flow chart of experiments in which GFP-positive <i>S</i>. <i>cerevisiae</i> was transformed with <i>S</i>. <i>castellii</i> or <i>E</i>. <i>histolytica</i> genes. Expression of the weak (Wk) silencing construct (long GFP dsRNA) or the strong (St) silencing construct (GFP hairpin) was induced in each strain. For each strain listed, high resolution small RNA Northern blots, total RNA Northern blots, and flow cytometry (FACS) analysis were performed. Northern blots and FACS histogram data shown refer to <i>S</i>. <i>cerevisiae</i> strains expressing <i>S</i>. <i>castellii</i> Dicer1 and <i>S</i>. <i>castellii</i> Ago1 and either the weak (Wk) or the strong (St) GFP silencing construct. These data recapitulate findings from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133740#pone.0133740.ref020" target="_blank">20</a>].</p

EhRNaseIII and EhAgo2-2 do not mediate silencing of GFP.

<p>GFP-positive <i>S</i>. <i>cerevisiae</i> untransformed (WT) and transformed with Myc-EhRNaseIII and EhAgo2-2 and expressing no GFP silencing construct (⦸), weak GFP long dsRNA silencing construct (Wk), or the strong GFP hairpin silencing construct (St). <b>(A)</b> Western blot from lysate of indicated strains probed with α-Myc, α-EhAgo2-2, or α-actin shows that EhRNaseIII and EhAgo2-2 proteins are expressed in all transformed cell lines. *: EhRNaseIII monomer; **: EhRNaseIII dimer. <b>(B)</b> High resolution Northern blot probed for GFP sRNAs in the strains listed in (A) indicates that EhRNaseIII does not generate abundant GFP sRNAs. <i>S</i>. <i>cerevisiae</i> expressing <i>S</i>. <i>castellii</i> Dicer1 and Ago1 serve as positive controls. Blot was stripped and re-probed for U6 small nucleolar RNAs as a loading control. <b>(C)</b> FACS histogram of GFP fluorescence in the indicated <i>S</i>. <i>cerevisiae</i> strains shows that EhRNaseIII and EhAgo2-2 are unable to silence GFP using either the weak or strong GFP silencing RNA substrates. <b>(D)</b> Upper: Northern blot probed for GFP mRNA in the indicated <i>S</i>. <i>cerevisiae</i> strains shows no decrease in GFP transcript in strains expressing EhRNaseIII and EhAgo2-2 and either the weak or strong GFP silencing construct. Strain DPB271 (no GFP) was used as negative control. Blot was stripped and re-probed for PYK1 as a loading control. Lower: Quantitation of GFP band intensities normalized by PYK1 expression. The corresponding values are relative to GFP expression in the absence of silencing constructs. Two experiments represented.</p