Zhang Hanbang, Ehrenkaufer Gretchen M, Hall Neil, Singh Upinder
Division of Infectious Diseases, Department of Internal Medicine, Stanford University School of Medicine, S-143 Grant Building, 300 Pasteur Drive, Stanford, CA, 94305-5107, USA.
Earlham Institute, Norwich Research Park, Norwich, NR4 7UH, UK.
BMC Genomics. 2020 Dec 9;21(1):879. doi: 10.1186/s12864-020-07275-6.
The RNA interference (RNAi) pathway is a gene regulation mechanism that utilizes 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 27 nt antisense sRNA populations which associate with EhAgo2-2 protein. However, there is lack of understanding about the sRNAs that are bound to two other EhAgos (EhAgo2-1 and 2-3), and the mechanism of sRNA regulation itself is unclear in this parasite. Therefore, identification of the entire pool of sRNA species and their sub-populations that associate with each individual EhAgo protein would be a major step forward.
In the present study, we sequenced sRNA libraries from both total RNAs and EhAgo bound RNAs. We identified a new population of 31 nt sRNAs that results from the addition of a non-templated 3-4 adenosine nucleotides at the 3'-end of the 27 nt sRNAs, indicating a non-templated RNA-tailing event in the parasite. The relative abundance of these two sRNA populations is linked to the efficacy of gene silencing for the target gene when parasites are transfected with an RNAi-trigger construct, indicating that non-templated sRNA-tailing likely play a role in sRNA regulation in this parasite. We found that both sRNA populations (27 nt and 31 nt) are present in the related parasite Entamoeba invadens, and are unchanged during the development. In sequencing the sRNAs associating with the three EhAgo proteins, we observed that despite distinct cellular localization, all three EhAgo sRNA libraries contain 27 nt sRNAs with 5'-polyphosphate (5'-polyP) structure and share a largely overlapping sRNA repertoire. In addition, our data showed that a fraction of 31 nt sRNAs associate with EhAgo2-2 but not with its mutant protein (C-terminal deletion), nor other two EhAgos, indicating a specific EhAgo site may be required for sRNA modification process in the parasite.
We identified a new population of sRNA with non-templated oligo-adenylation modification, which is the first such observation amongst single celled 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.
RNA干扰（RNAi）途径是一种基因调控机制，利用小RNA（sRNA）和AGO蛋白使靶基因沉默。我们之前的工作在原生动物寄生虫溶组织内阿米巴中鉴定出一种功能性RNAi途径，包括与EhAgo2-2蛋白相关的大量27nt反义sRNA群体。然而，对于与另外两种EhAgo（EhAgo2-1和2-3）结合的sRNA缺乏了解，并且在这种寄生虫中sRNA调控本身的机制尚不清楚。因此，鉴定与每个单独的EhAgo蛋白相关的sRNA种类及其亚群体的完整集合将是向前迈出的重要一步。
在本研究中，我们对总RNA和与EhAgo结合的RNA的sRNA文库进行了测序。我们鉴定出一种新的31nt sRNA群体，它是由27nt sRNA的3'末端添加非模板化的3 - 4个腺苷核苷酸产生的，这表明在该寄生虫中存在非模板化的RNA加尾事件。当用RNAi触发构建体转染寄生虫时，这两种sRNA群体的相对丰度与靶基因的基因沉默效率相关，表明非模板化的sRNA加尾可能在该寄生虫的sRNA调控中起作用。我们发现这两种sRNA群体（27nt和31nt）都存在于相关寄生虫侵袭内阿米巴中，并且在发育过程中保持不变。在对与三种EhAgo蛋白相关的sRNA进行测序时，我们观察到尽管细胞定位不同，但所有三个EhAgo sRNA文库都含有具有5'-多磷酸（5'-polyP）结构的27nt sRNA，并且共享很大程度上重叠的sRNA库。此外，我们的数据表明，一部分31nt sRNA与EhAgo2-2结合，但不与其突变蛋白（C末端缺失）或其他两种EhAgo结合，这表明在该寄生虫的sRNA修饰过程中可能需要特定的EhAgo位点。
我们鉴定出一种具有非模板化寡聚腺苷酸化修饰的新sRNA群体，这是在单细胞原生动物寄生虫中的首次此类观察。我们的sRNA测序文库为所有三种内阿米巴Ago蛋白提供了首个全面的sRNA数据集，可作为变形虫群落的有用数据库。
