Plant upstream ORFs can trigger nonsense-mediated mRNA decay in a size-dependent manner

Nonsense-mediated decay (NMD) is a quality control mechanism that identifies and degrades aberrant mRNAs containing premature termination codons (PTC). NMD also regulates the expression of many wild-type genes. In plants, NMD identifies a stop codon as a PTC and initiates the rapid degradation of the transcript if the 3'untranslated region (UTR) is unusually long or if it harbors an intron. Approximately 20% of plant transcripts have an upstream ORF (uORF) in the 5'UTR. In theory, if a uORF is translated, the 3'UTR downstream of the uORF will be long and harbor introns, thus these transcripts might be degraded by NMD. Therefore, if uORFs can trigger NMD, uORF containing transcripts would be a major group of NMD regulated wild-type plant mRNAs. The aim of this study was to clarify whether plant uORFs could activate NMD. Here we demonstrate that plant uORFs induce NMD in a size-dependent manner, a 50 amino acid (aa) long uORF triggered NMD efficiently, whereas similar but shorter (31 and 15 aa long) uORFs failed to activate NMD response. We have found that only similar to 2% of annotated Arabidopsis genes contain a first uORF that is longer than 35 aa, thus we propose that NMD regulates only a small fraction of uORF containing transcripts. However, as mRNAs having uORF that is longer than the critical size are strongly overrepresented within the up-regulated transcripts of NMD deficient plants, it is likely that this subset of natural NMD targets induces NMD because of containing a relatively long translatable uORF.