Division of Infectious Diseases, Department of Internal Medicine, Stanford University School of Medicine, Stanford, California 94305-5107, USA and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305-5107, USA.
Nucleic Acids Res. 2013 Nov;41(20):9424-37. doi: 10.1093/nar/gkt717. Epub 2013 Aug 9.
RNA interference uses small RNAs (sRNA), which target genes for sequence-specific silencing. The parasite Entamoeba histolytica contains an abundant repertoire of 27 nt antisense (AS) sRNA with 5'-polyphosphate termini, but their roles in regulating gene expression have not been well established. We demonstrate that a gene-coding region to which large numbers of AS sRNAs map can serve as a 'trigger' and silence the gene fused to it. Silencing is mediated by generation of AS sRNAs with 5'-polyphosphate termini that have sequence specificity to the fused gene. The mechanism of silencing is independent of the placement of the trigger relative to the silenced gene but is dependent on the sRNA concentration to the trigger. Silencing requires transcription of the trigger-gene fusion and is maintained despite loss of the trigger plasmid. We used this approach to silence multiple amebic genes, including an E. histolytica Myb gene, which is upregulated during oxidative stress response. Silencing of the EhMyb gene decreased parasite viability under oxidative stress conditions. Thus, we have developed a new tool for genetic manipulation in E. histolytica with many advantages over currently available technologies. Additionally, these data shed mechanistic insights into a eukaryotic RNA interference pathway with many novel aspects.
RNA 干扰利用小 RNA（sRNA），靶向基因进行序列特异性沉默。寄生虫溶组织内阿米巴含有丰富的 27 个核苷酸反义（AS）sRNA，其 5'-末端带有多聚磷酸基团，但它们在调节基因表达中的作用尚未得到很好的确立。我们证明，大量 AS sRNA 映射的基因编码区域可以作为“触发”，并沉默与其融合的基因。沉默是通过产生具有 5'-多聚磷酸基团的 AS sRNA 介导的，该 sRNA 对融合基因具有序列特异性。沉默机制独立于触发相对于沉默基因的位置，但依赖于触发处的 sRNA 浓度。沉默需要触发基因融合的转录，并且即使失去触发质粒也能维持。我们使用这种方法沉默了多个阿米巴基因，包括在氧化应激反应期间上调的溶组织内阿米巴 Myb 基因。EhMyb 基因沉默降低了寄生虫在氧化应激条件下的生存能力。因此，我们开发了一种新的溶组织内阿米巴遗传操作工具，具有许多优于现有技术的优点。此外，这些数据为具有许多新颖方面的真核 RNA 干扰途径提供了机制见解。
