Nonsense mutations can increase mRNA levels

Abstract Nonsense mutations are often associated with reduced mRNA levels, as premature translation termination can lead to the activation of nonsense-mediated mRNA decay (NMD). To examine how positional context influences these outcomes, we introduced premature translation termination codons (PTCs) at 15 locations within the coding region of a GFP reporter gene in Schizosaccharomyces pombe. PTCs in the first third of the coding region (codons 1–88) consistently led to reduced mRNA levels. In contrast, most downstream PTCs (codons 108–231) showed modest or minimal reductions, and several were associated with increased mRNA levels relative to the PTC-less control transcript. Measurement of transcript stability for one such variant indicated that the increased abundance was not attributable to decreased turnover. Deletion of UPF1 in wild-type cells elevated the levels of transcripts that were reduced and, unexpectedly, further increased the abundance of transcripts that exceeded the control level. In spliced versions of these constructs, downstream PTCs generally reduced mRNA levels regardless of exon junction position; additionally, for some early PTCs, splicing appeared to suppress rather than enhance mRNA reduction. Overall, these observations indicate that an unexpected consequence of nonsense mutations can be increased mRNA levels. These findings may aid in the interpretation of the effects of nonsense mutations on mRNA abundance beyond the predictions of current NMD models and may also help in the design of eukaryotic gene-expression constructs. Competing Interest Statement The authors have declared no competing interest. Footnotes In the current version, we have improved the readability of the text and the quality of the figures.