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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 .

plos one

High Throughput Sequencing of Entamoeba 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