Division of Infectious Diseases, Department of Internal Medicine, Stanford University School of Medicine, Stanford, California 94305-5107, USA.
BMC Genomics. 2013 Jan 25;14:53. doi: 10.1186/1471-2164-14-53.
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.
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.
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.
小 RNA 介导的基因沉默是一种保守的调控途径。在寄生虫溶组织内阿米巴中存在内源性 RNAi 途径，然而，小 RNA 群体的深度和多样性仍不清楚。
为了描述与溶组织内阿米巴 Argonaute-2（EhAGO2-2）相关的小 RNA 群体，我们免疫沉淀与之相关的小 RNA 并进行了一次完整的焦磷酸测序。数据分析揭示了 27nt 小 RNA 的新特征，包括 5'-G 优势、在蛋白质编码基因上的独特小 RNA 分布模式、映射到内含子和外显子-内含子交界处的小 RNA 以及小 RNA 靶向基因特别聚集在基因组复制区域。对小 RNA 映射到的基因组基因座的特征进行分析表明，正反义小 RNA 都具有 5'-多磷酸末端；在这些基因座上的链特异性 RT-PCR 检测到了两个方向的转录本，这表明这两个转录本都可能作为小 RNA 生成的模板。为了确定小 RNA 丰度模式是否解释了溶组织内阿米巴毒力和非毒力菌株的特定基因表达谱，我们对非毒力菌株的小 RNA 进行了测序，发现小 RNA 以与调节其特定毒力基因一致的方式映射到基因上。
我们对溶组织内阿米巴 EhAGO2-2 相关的 27nt 小 RNA 进行了全面分析。此外，对毒力和非毒力阿米巴菌株的小 RNA 群体进行比较分析表明，小 RNA 群体可能调节毒力基因。
