Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that recognizes and degrades transcripts containing NMD cis elements in their 3′untranslated region (UTR). In yeasts, unusually long 3′UTRs act as NMD cis elements, whereas in vertebrates, NMD is induced by introns located >50 nt downstream from the stop codon. In vertebrates, splicing leads to deposition of exon junction complex (EJC) onto the mRNA, and then 3′UTR-bound EJCs trigger NMD. It is proposed that this intron-based NMD is vertebrate specific, and it evolved to eliminate the misproducts of alternative splicing. Here, we provide evidence that similar EJC-mediated intron-based NMD functions in plants, suggesting that this type of NMD is evolutionary conserved. We demonstrate that in plants, like in vertebrates, introns located >50 nt from the stop induces NMD. We show that orthologs of all core EJC components are essential for intron-based plant NMD and that plant Partner of Y14 and mago (PYM) also acts as EJC disassembly factor. Moreover, we found that complex autoregulatory circuits control the activity of plant NMD. We demonstrate that expression of suppressor with morphogenic effect on genitalia (SMG)7, which is essential for long 3′UTR- and intron-based NMD, is regulated by both types of NMD, whereas expression of Barentsz EJC component is downregulated by intron-based NMD.
SUMMARYNonsense-mediated mRNA decay (NMD) is an essential quality control system that degrades aberrant transcripts containing premature termination codons and regulates the expression of several normal transcripts. Targets for NMD are selected during translational termination. If termination is slow, the UPF1 NMD factor binds the eRF3 protein of the termination complex and then recruits UPF2 and UPF3. Consequently, the UPF1-2-3 NMD complex induces SMG7-mediated degradation of the target mRNA. It is unknown how formation of the NMD complex and transcript degradation are linked in plants. Previously we have shown that the N-and C-terminal domains of UPF1 act redundantly and that the N-terminal domain is phosphorylated.To clarify the role of UPF1 phosphorylation in plant NMD, we generated UPF1 mutants and analyzed their phosphorylation status and the NMD competency of the mutants. We show that although several residues in the N-terminal domain of UPF1 are phosphorylated, only three phosphorylated amino acids, S3, S13 and T29, play a role in NMD. Moreover, we found that the C-terminal domain consists of redundant S/TQ-rich segments and that S1076 is involved in NMD. All NMD-relevant phosphorylation sites were in the S/TQ context. Co-localization and fluorescence resonance energy transfer-fluorescence lifetime imaging assays suggest that N-terminal and probably also C-terminal phosphorylated S/TQ residues are the binding platform for SMG7. Our data support the hypothesis that phosphorylation of UPF1 connects NMD complex formation and the SMG7-mediated target transcript degradation steps of NMD. SMG7 binds the phosphorylated S/TQ sites of the UPF1 component of the NMD complex, and then it induces the degradation of the NMD target.
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