The blots were sequentially reacted with primary anti bodies followed by horseradish peroxidase conjugated anti rabbit IgG antibodies according to manufacturers instructions. Chemi luminescence signals developed using ECL Plus kit. Some SKI-606 blots were stripped and re probed with anti ERK or p38 antibodies to determine equivalency of protein loading. The data from 3 4 repli cate experiments were quantified by densitometry, nor malized against total ERK or p38 or actin, and subjected to statistical analysis, as outlined previously. Background RNA interference is a well conserved gene re gulatory pathway found in most eukaryotes. Many important biological functions are controlled by RNAi such as developmental regulation, genome protection against viruses and transposons, and DNA elimin ation.

Small RNA molecules, usually 20 30nt, are the key elements for RNAi. Guided by their associated protein complexes, they base pair to the tar geted transcripts or genomic loci to trigger gene silen cing at either the transcriptional or post transcriptional Inhibitors,Modulators,Libraries level. In recent years, high throughput sequencing has facilitated the identification of diverse species of small Inhibitors,Modulators,Libraries RNAs in different organisms. Several protozoan parasites such as Trypanosoma bru cei, Toxoplasma gondii, Giardia lamblia, Trichomonas vaginalis, and Entamoeba histolytica contain key genes of the RNAi pathway in their genomes. The func tions of RNAi in parasite biology include retrotrans poson control in T. brucei, gene regulation in E. histolytica, and control of antigenic variation in G. lamblia. In T. gondii and T.

vaginalis the studies have been limited to endogenous small RNA sequencing with no functional studies yet reported. E. histolytica causes dysentery and liver abscesses Inhibitors,Modulators,Libraries in humans and affects 500 million people worldwide. The study of this important human parasite has been hampered by lack of standard molecular genetic tools due to the polyploid nature of the E. histolytica Inhibitors,Modulators,Libraries genome. Recently, several RNAi based gene knockdown ap proaches dsRNA/siRNA, short hairpin RNA and a transcriptional gene silencing Inhibitors,Modulators,Libraries approach in the G3 parasite strain have been established in this organism. We have shown that E. histolytica has a 27nt small RNA population, which has 50 polyphosphate and 30 OH termini and associates with EhAGO2 2. Additionally, we have demonstrated that gene si lencing in the E.

histolytica G3 strain is mediated through a siRNA product information pathway. The E. histolytica genome encodes three Argonaute proteins of which EhAGO2 2 is highly expressed and associates with 27nt small RNAs. In this report, we immunoprecipitated small RNAs bound to EhAGO2 2 and sequenced them using a high throughput pyrose quencing approach, which generated over 360,000 small RNA reads. Analysis of these endogenous small RNAs revealed that their peak length is 27nt and that there was a strong G bias at the 50 nucleotide.