Nonsense-mediated mRNA decay (NMD) is governed by the three conserved factors-UPF1, UPF2, and UPF3. While all three are required for NMD in yeast, UPF3B is dispensable for NMD in mammals, and its paralog UPF3A is suggested to only weakly activate or even repress NMD due to its weaker binding to the exon junction complex (EJC). Here, we characterize the UPF3A/B-dependence of NMD in human cell lines deleted of one or both UPF3 paralogs. We show that in human colorectal cancer HCT116 cells, NMD can operate in a UPF3B-dependent and -independent manner. While UPF3A is almost dispensable for NMD in wild-type cells, it strongly activates NMD in cells lacking UPF3B. Notably, NMD remains partially active in cells lacking both UPF3 paralogs. Complementation studies in these cells show that EJC-binding domain of UPF3 paralogs is dispensable for NMD. Instead, the conserved "mid" domain of UPF3 paralogs is consequential for their NMD activity. Altogether, our results demonstrate that the mammalian UPF3 proteins play a more active role in NMD than simply bridging the EJC and the UPF complex.

中文翻译：

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哺乳动物 UPF3A 和 UPF3B 可以独立于它们的外显子连接复合物结合激活无义介导的 mRNA 衰变。

无义介导的 mRNA 衰变 (NMD) 受三个保守因子 UPF1、UPF2 和 UPF3 控制。虽然酵母中的 NMD 需要这三者，但 UPF3B 对哺乳动物的 NMD 来说是可有可无的，并且由于其与外显子连接复合物 (EJC) 的结合较弱，建议其旁系同源 UPF3A 仅弱激活甚至抑制 NMD。在这里，我们描述了 NMD 在删除一个或两个 UPF3 旁系同源物的人类细胞系中的 UPF3A/B 依赖性。我们表明，在人类结直肠癌 HCT116 细胞中，NMD 可以以 UPF3B 依赖性和非依赖性方式运作。虽然 UPF3A 对于野生型细胞中的 NMD 几乎是可有可无的，但它强烈激活缺乏 UPF3B 的细胞中的 NMD。值得注意的是，NMD 在缺乏 UPF3 旁系同源物的细胞中仍然部分活跃。这些细胞中的互补研究表明，UPF3 旁系同源物的 EJC 结合域对于 NMD 是可有可无的。反而，UPF3 旁系同源物的保守“中间”结构域对其 NMD 活性至关重要。总之，我们的研究结果表明，哺乳动物 UPF3 蛋白在 NMD 中发挥的作用比简单地桥接 EJC 和 UPF 复合物更积极。