Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic RNA surveillance mechanism that degrades aberrant mRNAs comprising a premature translation termination codon. The adenosine triphosphate (ATP)-dependent RNA helicase up-frameshift 1 (UPF1) is a major NMD factor in all studied organisms; however, the complexity of this mechanism has not been fully characterized in plants. To identify plant NMD factors, we analyzed UPF1-interacting proteins using tandem affinity purification coupled to mass spectrometry. Canonical members of the NMD pathway were found along with numerous NMD candidate factors, including conserved DEA(D/H)-box RNA helicase homologs of human DDX3, DDX5 and DDX6, translation initiation factors, ribosomal proteins and transport factors. Our functional studies revealed that depletion of DDX3 helicases enhances the accumulation of NMD target reporter mRNAs but does not result in increased protein levels. In contrast, silencing of DDX6 group leads to decreased accumulation of the NMD substrate. The inhibitory effect of DDX6-like helicases on NMD was confirmed by transient overexpression of RH12 helicase. These results indicate that DDX3 and DDX6 helicases in plants have a direct and opposing contribution to NMD and act as functional NMD factors.

中文翻译：

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DEA(D/H)-Box 家族的 RNA 解旋酶有助于植物 NMD 效率。


无义介导的 mRNA 降解 (NMD) 是一种保守的真核 RNA 监视机制，可降解包含过早翻译终止密码子的异常 mRNA。三磷酸腺苷 (ATP) 依赖性 RNA 解旋酶上移码 1 (UPF1) 是所有研究生物体中的主要 NMD 因子；然而，这种机制的复杂性尚未在植物中得到充分表征。为了鉴定植物 NMD 因子，我们使用串联亲和纯化结合质谱分析了 UPF1 相互作用蛋白。发现了 NMD 途径的典型成员以及许多 NMD 候选因子，包括人 DDX3、DDX5 和 DDX6 的保守 DEA(D/H)-box RNA 解旋酶同源物、翻译起始因子、核糖体蛋白和转运因子。我们的功能研究表明，DDX3 解旋酶的消耗会增强 NMD 靶标报告基因 mRNA 的积累，但不会导致蛋白质水平增加。相反，DDX6 基团的沉默导致 NMD 底物的积累减少。 DDX6 样解旋酶对 NMD 的抑制作用通过 RH12 解旋酶的瞬时过表达得到证实。这些结果表明植物中的 DDX3 和 DDX6 解旋酶对 NMD 具有直接和相反的贡献，并充当功能性 NMD 因子。