Position-Dependent NMD Generates Diverse Protein Outcomes

Nonsense-mediated mRNA decay (NMD) is a translation-dependent mRNA decay pathway triggered by premature termination codons (PTCs). Although NMD is known to eliminate aberrant transcripts, how PTC position influences cell-to-cell heterogeneity in NMD and the resulting protein outputs remains unclear. Here, we used a single-cell NMD analysis system that quantifies cellular variability based on the GFP/mCherry fluorescence ratio. By combining this system with fluorescence-activated cell sorting (FACS), we show that PTC location critically determines not only NMD efficiency and its variability across cells, but also the spectrum of resulting protein products. These include truncated proteins arising from premature termination, full-length proteins generated through translational readthrough, and N-terminally truncated isoforms produced by downstream reinitiation. Our findings reveal that positional and cellular heterogeneity in NMD contribute to proteomic diversity and may underlie the variable phenotypic severity of genetic diseases caused by PTCs. This work establishes a framework for dissecting NMD regulation and its translational consequences.