Nucleus-localized Antisense Small RNAs with 5′-Polyphosphate Termini Regulate Long Term Transcriptional Gene Silencing in Entamoeba histolytica G3 Strain

Abstract

Background: The mechanism(s) of G3-based TGS in E. histolytica is largely unknown. Results: 5Ј-polyphosphate antisense sRNAs are identified; mechanistic insights linking these sRNAs with TGS are provided by IFA, FISH, IP, and ChIP assays. Conclusion: TGS in E. histolytica G3 strain is mediated by an siRNA pathway, which utilizes antisense 5Ј-polyphosphate sRNAs. Significance: This is the first demonstration of (a) 5Ј-polyphosphate antisense sRNAs mediating TGS and (b) RNAi-mediated TGS in protozoan parasites.

Dimethylated H3K27 Is a Repressive Epigenetic Histone Mark in the Protist Entamoeba histolytica and Is Significantly Enriched in Genes Silenced via the RNAi Pathway

“…Previously, using a commercial ␣-H3 antibody we and others have demonstrated increased occupancy of H3 at loci affected by small RNA (sRNA)-mediated gene silencing in the E. histolytica G3 strain (33,37). To more specifically investigate the mechanism of small RNA-induced transcriptional gene silencing, we asked if post-translationally modified H3 histone (H3K27Me2) contributes to small RNA gene silencing in E. histolytica.…”

“…Although many genes could be silenced using this approach, we also identified some genes that could not be silenced despite the generation of functional small RNAs (36). It has previously been demonstrated that RNAi-silenced loci have increased histone occupancy, however, given the sequence divergence of the amebic H3, commercially available reagents could not be applied to define the molecular modifications typically associated with heterochromatin (33,37).…”

“…The percentage of dimethylated Lys-27 in H3K27Me2 peptide preparations was 99%. Anti-EhAgo2-2 and anti-Eh-RBP9 (or anti-Pol II) antibodies were generated in previous studies (33,38). Commercial antibodies included a pan-specific mouse anti-actin antibody (MP Biomedicals, Burlingame, CA); secondary HRP-conjugated anti-mouse and anti-rabbit antibodies (Jackson ImmunoResearch, West Grove, PA) were also utilized.…”

“…Immunofluorescence Microscopy-Fluorescence microscopy was performed as described previously (33). Wild type E. histolytica trophozoites were grown for 48 h. Confluent cells were allowed to adhere onto a glass slide chamber (Thermo Fisher) for 3 h at 37°C in TYI-S-33 medium.…”

“…Despite its position as a deep-branching eukaryote, E. histolytica has a distinct and complex endogenous RNAi pathway mediated by 27-nucleotide 5Ј-poly-P small RNAs that map antisense to genes and mediate TGS via a nuclear-localized Argonaute protein (33,34). We have recently developed a robust silencing tool in ameba by harnessing the endogenous RNAi pathway.…”