Small RNA pyrosequencing in the protozoan parasite Entamoeba histolytica reveals strain-specific small RNAs that target virulence genes

Hanbang Zhang,Upinder Singh,Gretchen M Ehrenkaufer,Neil Hall

doi:10.1186/1471-2164-14-53

Hanbang Zhang, Upinder Singh + Show 2 more

Open Access

https://doi.org/10.1186/1471-2164-14-53

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Abstract

BackgroundSmall RNA mediated gene silencing is a well-conserved regulatory pathway. In the parasite Entamoeba histolytica an endogenous RNAi pathway exists, however, the depth and diversity of the small RNA population remains unknown.ResultsTo characterize the small RNA population that associates with E. histolytica Argonaute-2 (EhAGO2-2), we immunoprecipitated small RNAs that associate with it and performed one full pyrosequencing run. Data analysis revealed new features of the 27nt small RNAs including the 5′-G predominance, distinct small RNA distribution patterns on protein coding genes, small RNAs mapping to both introns and exon-exon junctions, and small RNA targeted genes that are clustered particularly in sections of genome duplication. Characterization of genomic loci to which both sense and antisense small RNAs mapped showed that both sets of small RNAs have 5′-polyphosphate termini; strand-specific RT-PCR detected transcripts in both directions at these loci suggesting that both transcripts may serve as template for small RNA generation. In order to determine whether small RNA abundance patterns account for strain-specific gene expression profiles of E. histolytica virulent and non-virulent strains, we sequenced small RNAs from a non-virulent strain and found that small RNAs mapped to genes in a manner consistent with their regulation of strain-specific virulence genes.ConclusionsWe provided a full spectrum analysis for E. histolytica AGO2-2 associated 27nt small RNAs. Additionally, comparative analysis of small RNA populations from virulent and non-virulent amebic strains indicates that small RNA populations may regulate virulence genes.

Highlights

Small RNA mediated gene silencing is a well-conserved regulatory pathway
High-throughput pyrophosphate sequencing of EhAGO22-bound small RNAs A library of EhAGO2-2 associated small RNAs was generated from RNA obtained by immunoprecipitation (IP) with anti-Myc antibody in E. histolytica HM-1:IMSS cells stably expressing Myc-tagged EhAGO2-2
We have previously shown that EhAGO2-2 associates with 27nt small RNAs, which have 50-polyP and 30-OH termini [11]

Summary

Introduction

In the parasite Entamoeba histolytica an endogenous RNAi pathway exists, the depth and diversity of the small RNA population remains unknown. RNA interference (RNAi) is a well-conserved gene regulatory pathway found in most eukaryotes [1,2]. Small RNA (sRNA) molecules, usually 20 – 30nt, are the key elements for RNAi. Guided by their. Several protozoan parasites such as Trypanosoma brucei, Toxoplasma gondii, Giardia lamblia, Trichomonas vaginalis, and Entamoeba histolytica contain key genes of the RNAi pathway in their genomes [8,9]. The functions of RNAi in parasite biology include retrotransposon control in T. brucei [10], gene regulation in E. histolytica [11,12], and control of antigenic variation in. In T. gondii and T. vaginalis the studies have been limited to endogenous small RNA sequencing with no functional studies yet reported [15,16]

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