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• W4248288071 abstract "Abstract Background: The RNA interference (RNAi) pathway is a gene regulation mechanism that uitilizes small RNA (sRNA) and Argonaute (Ago) proteins to silence target genes. Our previous work identified a functional RNAi pathway in the protozoan parasite Entamoeba histolytica , including abundant 27nt antisense sRNA populations derived from the secondary RNAi pathway which associate with Eh Ago2-2 protein. However, there is lack of understanding about sRNAs that are bound to two other Eh Agos (Eh Ago2-1 and 2-3), and the mechanism of sRNA regulation itself is unclear in this parasite. Results: In the present study, we sequenced sRNA libraries from both total RNAs and Eh Ago bound RNAs. We identified a new population of 31nt sRNAs that results from the addition of a non-templated 3-4 adenosine nucleotides at the 3´-end of the 27nt sRNA populations, indicating a non-templated RNA-tailing event in the parasite. We found that both sRNA populations (27nt and 31nt) are unchanged during the development of E. invadens . However, we detected an alteration in their relative abundance for the targeted gene in parasites transfected with a trigger-gene silencing construct, indicating that non-templated RNA-tailing is likely a pathway for sRNA turnover when the targeted gene is unable to be silenced in this parasite. In sequencing the sRNAs associating with the three Eh Ago proteins, we observed that despite distinct cellular localization, all three Eh Ago sRNA libraries contain 27nt sRNAs with 5´-polyphosphate (5´-polyP) structure and a largely overlapping sRNA repertoire, mainly targeting retrotransposons and a subset of ~226 genes that are endogenously silenced. Furthermore, our data show that 31nt sRNA populations paritally associate with wildtype Eh Ago2-2 but not with its mutant protein ( Eh Ago2-2 C-terminal deletion), indicating an intact RISC is essential for the sRNA modification process. Conclusion: High-throughput sequencing of sRNA in Entamoeba has identified a new population of sRNA with non-templated adenylation modification, which is the first such observation amongst single cell protozoan parasites. Our sRNA sequencing libraries provide the first comprehensive sRNA dataset for all three Entamoeba Ago proteins, which can serve as a useful database for the amoeba community." @default.
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• W4248288071 date "2020-10-05" @default.
• W4248288071 modified "2023-09-29" @default.
• W4248288071 title "Non-Templated Oligo-Adenylation of Small RNAs in the Parasite Entamoeba: Potential Roles for Small RNA Control in Single Celled Eukaryotic Pathogen" @default.
• W4248288071 doi "https://doi.org/10.21203/rs.3.rs-41465/v1" @default.
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