Abstract
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.
Highlights
Nonsense-mediated mRNA decay (NMD) is an mRNA degradation pathway that eliminates transcripts containing premature termination codons (PTCs)
“faux 3’untranslated region (UTR)”, proposes that the distance from the termination codon to the poly (A) tail provides for the targeting of NMD due to the competitive binding of the major NMD regulator, up-frameshift factor 1 (UPF1), and cytoplasmic poly (A) binding protein 1 (PABC1) with eukaryotic polypeptide chain release factor 3, which interacts with eRF1 and recognizes termination codons including the PTC6
The results showed that NMD efficiency was correlated with the level of SMG1 (Fig. 3a, b, c); less correlation was found with the level of UPF1 or with SMG6 (Fig. S7)
Summary
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. The ordinary approaches to determine NMD efficiency, characterizing half-lives of PTC-containing transcripts compared with normal transcripts are generally ensemble assays. These determine the mean of NMD efficiency but are unable to investigate the heterogeneities in the population with single-cell resolution
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