• 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.

中文翻译：

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拟南芥ABCE2的突变分析确定了其RNA沉默抑制功能的重要基序

• 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循环因子，在古细菌和真核生物中翻译是必不可少的，然而，它在植物中的作用在很大程度上仍未确定。拟南芥编码两种旁系同源 ABCE 蛋白（AtABCE1 和 AtABCE2），具有 81% 的同一性。我们之前报道过 AtABCE2 可作为 RNA 沉默的抑制因子，并且其基因普遍表达。在这里，我们描述了 AtABCE2 对其抑制功能的结构要求。
• 使用农杆菌浸润测定法，我们将 AtABCE2 的突变版本与 GFP 一起瞬时过表达，以在 GFP 转基因本氏烟草叶中诱导沉默。通过 GFP的体内成像、GFP siRNA 的 Northern 印迹分析和在紫外线下观察植物，在局部和全身水平上分析了突变的影响。
• AtABCE2 在活性位点 I 或 II 中的 ATP 结合受损的突变体未能抑制 GFP RNA 沉默。破坏 ATP 水解的突变对活性位点 I 或 II 的沉默抑制有不同的影响。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。
• 有意义的是，发现 AtABCE2 对 RNA 沉默抑制的结构要求与核糖体回收所需的古细菌 ABCE1 的结构要求相似。因此，AtABCE2 可能通过支持与核糖体循环相关的竞争性 RNA 降解机制来抑制 RNA 沉默。