Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, Estonia.
Plant Biol (Stuttg). 2021 Jan;23(1):21-31. doi: 10.1111/plb.13193. Epub 2020 Nov 29.
ATP-binding cassette sub-family E member 1 (ABCE1) is recognized as a strongly conserved ribosome recycling factor, indispensable for translation in archaea and eukaryotes, however, its role in plants remains largely unidentified. Arabidopsis thaliana encodes two paralogous ABCE proteins (AtABCE1 and AtABCE2), sharing 81% identity. We previously reported that AtABCE2 functions as a suppressor of RNA silencing and that its gene is ubiquitously expressed. Here we describe the structural requirements of AtABCE2 for its suppressor function. Using agroinfiltration assays, we transiently overexpressed mutated versions of AtABCE2 together with GFP, to induce silencing in GFP transgenic Nicotiana benthamiana leaves. The influence of mutations was analysed at both local and systemic levels by in vivo imaging of GFP, Northern blot analysis of GFP siRNAs and observation of plants under UV light. Mutants of AtABCE2 with impaired ATP binding in either active site I or II failed to suppress GFP RNA silencing. Mutations disrupting ATP hydrolysis influenced the suppression of silencing differently at active site I or II. We also found that the N-terminal iron-sulphur cluster domain of AtABCE2 is crucial for its suppressor function. Meaningfully, the observed structural requirements of AtABCE2 for RNA silencing suppression were found to be similar to those of archaeal ABCE1 needed for ribosome recycling. AtABCE2 might therefore suppress RNA silencing via supporting the competing RNA degradation mechanisms associated with ribosome recycling.
ATP 结合盒亚家族 E 成员 1（ABCE1）被认为是一种高度保守的核糖体回收因子，对于古菌和真核生物的翻译是必不可少的，但它在植物中的作用在很大程度上仍未被确定。拟南芥编码两个同源的 ABCE 蛋白（AtABCE1 和 AtABCE2），它们具有 81%的同源性。我们之前报道过 AtABCE2 作为 RNA 沉默的抑制剂发挥作用，并且其基因在广泛表达。在这里，我们描述了 AtABCE2 发挥其抑制功能的结构要求。我们使用农杆菌浸润实验，瞬时过表达 GFP 与 AtABCE2 的突变体，以诱导 GFP 转基因烟草叶片中的沉默。通过 GFP 的体内成像、GFP siRNAs 的 Northern blot 分析以及在紫外线下观察植物，分析了突变在局部和系统水平上的影响。在任一活性位点 I 或 II 中，ATP 结合能力受损的 AtABCE2 突变体无法抑制 GFP RNA 沉默。在活性位点 I 或 II 上破坏 ATP 水解的突变以不同的方式影响沉默的抑制。我们还发现 AtABCE2 的 N 端铁硫簇结构域对其抑制功能至关重要。有意思的是，AtABCE2 抑制 RNA 沉默所需的结构要求与古菌 ABCE1 用于核糖体回收的要求相似。因此，AtABCE2 可能通过支持与核糖体回收相关的竞争性 RNA 降解机制来抑制 RNA 沉默。
