Nonsense-mediated mRNA decay (NMD) silences the accumulation of aberrant trypsin proteinase inhibitor mRNA in Nicotiana attenuata

In eukaryotes, genes carrying premature termination codons (PTCs) are often associated with decreased mRNA levels compared with their counterparts without PTCs. PTC-harboring mRNA is rapidly degraded through the nonsense-mediated mRNA decay (NMD) pathway to prevent the accumulation of potentially detrimental truncated proteins. In a native ecotype of Nicotiana attenuata collected from Arizona (AZ), the mRNA levels of a trypsin proteinase inhibitor (TPI) gene are substantially lower than in plants collected from Utah (UT). Cloning the AZ TPI gene revealed a 6 bp deletion mutation in exon 2 resulting in a PTC and decreased mRNA levels through NMD. Silencing UPF1, 2 and 3 in N. aftenuata AZ plants by virus-induced gene silencing (VIGS) enhanced the levels of PTC-harboring TP1 mRNA, demonstrating a conserved role for UPF genes in plants. Furthermore, using cell suspension cultures that express variants of the TPI construct, we demonstrate that both intron-containing and intronless genes are subject to NIVID in plants; unlike PTCs in mammals, PTCs downstream of introns activate NIVID in plants. However, when a PTC is only 4 bp upstream of an intron, the NIVID surveillance mechanism is abrogated. We also demonstrate that, in an intronless TPI gene, a PTC located at the beginning or the end of the coding sequence triggers NIVID less efficiently than do PTCs located at the middle of the coding sequence. Taken together, these results highlight the complexity of the NIVID activation mechanisms in plants.