High Throughput Sequencing of <i>Entamoeba</i> 27nt Small RNA Population Reveals Role in Permanent Gene Silencing But No Effect on Regulating Gene Expression Changes during Stage Conversion, Oxidative, or Heat Shock Stress

The human parasite Entamoeba histolytica has an active RNA interference (RNAi) pathway with an extensive repertoire of 27nt small RNAs that silence genes. However the role of this pathway in regulating amebic biology remains unknown. In this study, we address whether silencing via 27nt small RNAs may be a mechanism for controlling gene expression changes during conversion between the trophozoite and cyst stages of the parasite. We sequenced small RNA libraries generated from trophozoites, early cysts, mature cysts, and excysting cells and mapped them to the E. invadens genome. Our results show that, as in E. histolytica, small RNAs in E. invadens are largely ~27nt in length, have an unusual 5'-polyphosphate structure and mediate gene silencing. However, when comparing the libraries from each developmental time-point we found few changes in the composition of the small RNA populations. Furthermore, genes targeted by small RNAs were permanently silenced with no changes in transcript abundance during development. Thus, the E. invadens 27nt small RNA population does not mediate gene expression changes during development. In order to assess the generalizability of our observations, we examined whether small RNAs may be regulating gene expression changes during stress response in E. histolytica. Comparison of the 27nt small RNA populations from E. histolytica trophozoites from basal conditions, or after heat shock or exposure to oxidative stress showed few differences. Similar to data in E. invadens development, genes targeted by small RNAs were consistently silenced and did not change expression under tested stress conditions. Thus, the biological roles of the 27nt small RNA population in Entamoeba remain elusive. However, as the first characterization of the RNAi pathway in E. invadens these data serve as a useful resource for the study of Entamoeba development and open the door to the development of RNAi-based gene silencing tools in E. invadens.溶组织内阿米巴（Entamoeba histolytica）拥有活跃的RNA干扰（RNA interference, RNAi）通路，其包含丰富的27nt小RNA库，可介导基因沉默。然而，该通路在调控阿米巴生物学特性中的功能仍未明确。本研究旨在探究27nt小RNA介导的基因沉默是否为寄生虫滋养体（trophozoite）与包囊（cyst）阶段互变过程中调控基因表达变化的潜在机制。我们对来源于滋养体、早期包囊、成熟包囊以及脱囊细胞的小RNA文库进行测序，并将测序序列比对至侵袭内阿米巴（E. invadens）基因组。结果显示，与溶组织内阿米巴类似，侵袭内阿米巴的小RNA长度大多约为27nt，具备独特的5'多磷酸结构，且可介导基因沉默。但对比各发育时间点的文库，我们发现小RNA群体的组成几乎无显著变化。此外，被小RNA靶向的基因始终处于沉默状态，发育过程中转录本丰度未发生任何改变。由此可见，侵袭内阿米巴的27nt小RNA群体并未介导发育过程中的基因表达变化。为评估本研究结果的普适性，我们进一步探究了溶组织内阿米巴在应激反应过程中，小RNA是否参与调控基因表达变化。对比基础培养条件、热休克（heat shock）处理或氧化应激（oxidative stress）处理后的溶组织内阿米巴滋养体的27nt小RNA群体，未发现显著差异。与侵袭内阿米巴发育阶段的研究结果一致，被小RNA靶向的基因始终处于沉默状态，在受试应激条件下其表达水平未发生改变。因此，阿米巴属27nt小RNA群体的生物学功能仍有待阐明。然而，作为首次对侵袭内阿米巴RNAi通路的特征性研究，本研究的数据可为阿米巴属发育研究提供宝贵资源，并为在侵袭内阿米巴中开发基于RNA干扰的基因沉默工具开辟了新路径。