Signal amplification is controlled by using Dorsomorphin ALK primary trigger siRNAs to instigate secondary siRNAs through RdRP for the enforced silencing, but limiting secondary siRNAs from doing further signal amplification. Although small RNAs that mapped sense to coding regions were found in C. elegans and As caris suum 5 monoP independent libraries, their exist ence and functionality were not confirmed. instead they were generally treated as non specific degradation pro ducts. We have confirmed that in E. histolytica small RNAs that map sense to genes are detected in Northern blot analyses as discrete band and bear the same 50 polyP ter mini. Strand specific RT PCRs detected transcripts in both directions for these loci, implying that RdRP based small RNA generation could occur for both sense and antisense transcripts in this parasite.
The E. histolytica genome encodes one full RdRP gene and two genes with partial RdRP domains. The functions of the RdRP genes in E. his tolytica RNAi pathway are still elusive and need further investigation. Due to the fact that gene knockout is not feasible in E. histolytica we have been unable to dissect how the para site RNAi components affect the levels Inhibitors,Modulators,Libraries of these small RNAs. The comparison of 50 polyP small RNAs among the three organisms in which they have been described show several dif ferences 50 polyP small RNA size in E. histolytica is 27nt, whereas in C. elegans and Ascaris these RNAs are 22nt. the distribution pattern of antisense small RNAs to the targeted gene loci is enriched at the 50 end for E. histolytica and Ascaris whereas in C.
elegans there is enrichment at the 30 end of transcripts. localization of EhAGO2 2 and bound 27nt small RNAs are mostly localized to the parasite nucleus, whereas C. elegans 22G Inhibitors,Modulators,Libraries RNAs Inhibitors,Modulators,Libraries can associate with several different WAGOs and have both perinuclear and nuclear localization . and C. elegans strongly prefers spliced tran scripts as RdRP template for Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries generating 50 polyP small RNAs whereas both mature and nascent transcripts ap pear to function as templates in E. histolytica. Future studies aimed at elucidating these different mechanisms are needed. Our previous limited Sanger sequencing has shown that small RNAs in E. histolytica largely mapped to the coding genes. Our pyrosequencing data further con firmed this mapping, which is in contrast to other pa rasitic systems T. gondii, G. intestinalis and T. brucei where the non-small-cell lung carcinoma small RNAs are 50 single phosphate and mostly derived from repetitive elements, retrotranspo sons. The genome of E. histolytica contains hundreds of copies of LINE and SINE elements, with SINE elements actively transcribed and LINE1 transcript detected by Northern blot analysis. How retro transposons are controlled in ameba is not known.