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Open Access Highly Accessed Research article

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

Hanbang Zhang1, Gretchen M Ehrenkaufer1, Neil Hall2 and Upinder Singh134*

Author affiliations

1 Division of Infectious Diseases, Department of Internal Medicine, Stanford University School of Medicine, Stanford, California, 94305-5107, USA

2 School of Biological Sciences, Biosciences Building, University of Liverpool, Crown Street, Liverpool, L69 7ZB, UK

3 Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, 94305-5107, USA

4 Department of Medicine, Division of Infectious Diseases, S-143 Grant Building, 300 Pasteur Drive, Stanford, CA, 94305, USA

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Citation and License

BMC Genomics 2013, 14:53  doi:10.1186/1471-2164-14-53

Published: 25 January 2013

Abstract

Background

Small 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.

Results

To 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.

Conclusions

We 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.

Keywords:
5-Polyphosphate small RNA; Argonaute; High-throughput pyrosequencing; Parasite