Nonsense mutations can also increase mRNA levels

Nonsense mutations are frequently associated with reduced mRNA levels due to premature translation termination triggering nonsense-mediated mRNA decay (NMD). We introduced premature termination codons (PTCs) at 15 different positions within the coding region of a GFP reporter gene in Schizosaccharomyces pombe. Mutations located in the first third of the coding region typically led to decreased mRNA levels, whilst those occurring further downstream caused either no change or only minor reductions, consistent with current splicing-independent NMD models. However, PTCs located closer to the normal stop codon unexpectedly resulted in increased mRNA levels compared to the PTC-less control transcript. Analysis of intron-containing versions of these constructs confirmed that splicing enhances the impact of a nonsense mutation on mRNA levels. As expected, deletion of UPF1 in wild-type cells increased the levels of transcripts normally reduced by NMD. Interestingly, it also further increased the levels of transcripts that were already elevated compared to the PTC-less control in the wild-type background. The greater abundance of these mRNAs does not appear to be due to increased mRNA stability. Notably, a PTC located near the end of the coding sequence not only resulted in increased mRNA levels, but also in the production of a truncated, yet functional, GFP protein. These observations challenges current key assumptions about the significance of NMD.