Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY, 10461, USA.
Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY, 10461, USA.
Nat Commun. 2021 Dec 10;12(1):7203. doi: 10.1038/s41467-021-27423-0.
Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that eliminates transcripts containing premature termination codons (PTCs). Half-lives of the mRNAs containing PTCs demonstrate that a small percent escape surveillance and do not degrade. It is not known whether this escape represents variable mRNA degradation within cells or, alternatively cells within the population are resistant. Here we demonstrate a single-cell approach with a bi-directional reporter, which expresses two β-globin genes with or without a PTC in the same cell, to characterize the efficiency of NMD in individual cells. We found a broad range of NMD efficiency in the population; some cells degraded essentially all of the mRNAs, while others escaped NMD almost completely. Characterization of NMD efficiency together with NMD regulators in single cells showed cell-to-cell variability of NMD reflects the differential level of surveillance factors, SMG1 and phosphorylated UPF1. A single-cell fluorescent reporter system that enabled detection of NMD using flow cytometry revealed that this escape occurred either by translational readthrough at the PTC or by a failure of mRNA degradation after successful translation termination at the PTC.
无意义介导的 mRNA 降解（NMD）是一种 mRNA 降解途径，可消除含有提前终止密码子（PTC）的转录本。含有 PTC 的 mRNA 的半衰期表明，一小部分逃避了监控而不会降解。目前尚不清楚这种逃避是代表细胞内 mRNA 降解的可变性，还是细胞群体对其具有抗性。在这里，我们通过双向报告基因的单细胞方法，在同一细胞中表达带有或不带有 PTC 的两个β-珠蛋白基因，以表征单个细胞中 NMD 的效率。我们发现群体中 NMD 效率的范围很广；一些细胞降解了几乎所有的 mRNA，而另一些细胞则几乎完全逃避了 NMD。在单细胞中对 NMD 效率和 NMD 调节剂进行表征表明，NMD 的细胞间可变性反映了监控因子 SMG1 和磷酸化 UPF1 的差异水平。一种单细胞荧光报告基因系统，通过流式细胞术检测 NMD 的发生，表明这种逃避是通过 PTC 的翻译通读或在 PTC 成功翻译终止后 mRNA 降解失败而发生的。
