Plant nonsense-mediated mRNA decay is controlled by different autoregulatory circuits and can be induced by an EJC-like complex

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.

To clarify whether the putative EJC components are required for plant NMD, VIGS vectors containing a segment from one of the EJC component were generated. To clone TRV-P-Barentsz (TRV-P-Ba.) and TRV-P-4A3 clones, ~600 nt long exonic sequences were amplified from N. benthamiana cDNA with NbBar VIGSF/NbBar VIGSR and with Nb4A3 VIGS F/Nb4A3VIGS R primer pairs. The PCR fragments were cleaved and cloned into EcoRI digested TRV-PDS vector.
To analyse the role of PYM in plant NMD, agroinfiltration constructs expressing the full length PYM protein or its C-terminal or N-terminal deletion versions were created. To clone the PYM construct, an RT-PCR product generated from N. tabaccum cDNA with Nt PYM F and Nt PYM R primers was cleaved with BamHI/EcoRI, and then the digested PCR fragment was cloned into pBIN-Sanyi vector. PYM-N and PYM-C constructs were generated from PYM with Nt PYM F/Nt PYM N R and with Nt PYM C F/ Nt PYM R primer pairs. The PCR fragments were digested and cloned into BamHI/EcoRI cleaved pBIN-Sanyi vector.
To clarify whether the 3'UTRs of Barentsz 1 and SMG7 transcripts can induce NMD, Barentsz 1 and SMG7 terminator test constructs were generated. To create G-B 1 T construct Arabidopsis Barentsz 1 (At1g80000) terminator region (the sequence from the stop to ~200 nt downstream from the polyadenylation site) was PCR amplified from genomic DNA using B1-T-F /B1-T-R primer pairs, and then the PCR fragment was cloned into a BamHI/EcoRI cleaved Bin-GFP vector. To generate G-B 1 nospli.T, an unsplicable mutant version of the G-B1T construct in which the splicing acceptor site is impaired, PCR mutagenesis was performed using B1-T-F/ B1-T-3'ss mutR G-B1 and B1-T-3'ss mutF/B1-T-R primer pairs. The mutagenized PCR fragment was cleaved and cloned into BamHI/EcoRI digested Bin-GFP vector.
To study the role of the two 3'UTR introns in the SMG7 NMD regulation, mutants lacking intron 6 (G-S7Noi.6T) or intron 7 (G-S7Noi.7T) were generated by PCR mutagenesis from the G-S7T construct. To generate mutated PCR fragment lacking intron 6, G-F/SMG7-T-6idel-R and SMG7-T-6idel-F/SMG7-T-R primer pairs were used. To create mutant PCR product lacking intron 7, G-F/SMG7-T-7idel-R and SMG7-T-7idel-F/SMG7-T-R primer pairs were used. To generate a construct lacking both introns (G-S7Noi.T) PCR mutagenesis was carried out on G-S7Noi.6T construct using G-F/SMG7-T-7idel-R and SMG7-T-7idel-F/SMG7-T-R primers. The three mutant PCR fragments were cleaved and cloned into  Ubiquitin (At5g25760, Ubi) was used as a control for qRT-PCR studies,    schematic, non-proportional representation of the reporter constructs that were used for protoplast transfection assays. Protoplast isolated from the leaves of wild-type (w-t) and NMD mutant (upf1-5 and upf3-1) Arabidopsis plants were co-transfected with two plasmids, a normalization control plasmid expressing luciferase (luc) transcript and a plasmid that expressed the G-95 control mRNA or the G-600 long NMD reporter or the G-95I intronbased NMD reporter transcripts. RNA gel blot assays were carried out from total RNAs isolated at 1 d.p.i. After normalization the G95 value was taken as 1. upf1-5 is a hypomorphic mutant, while upf3-1 might be a null mutant. In w-t control protoplasts, both NMD reporter transcript accumulate to low levels relative to the G-95 control, indicating that both types of NMD function in Arabidopsis leaf protoplast. Note that in upf1-5 mutant the control G-95 and the G-600 and G-95I NMD reporter transcripts express to comparable levels. By contrast, in upf3-1 mutant protoplasts the G-600 mRNA accumulates to relatively low levels, while the G-95I reporter and the G-95 control transcripts accumulate to comparable levels.
These data indicate that UPF1 is essential for both types of NMD and that UPF3 is required for intron-based NMD. Apparently, UPF3 enhances the efficacy of long 3'UTR-based NMD but it is not essential for long 3'UTR-based NMD. (B) Schematic, non-proportional representation of the constructs that were used for VIGS-agroinfiltration assay. TRV-P, TRV-P-U2 and TRV-P-S7 VIGS construct were used to silence the control (PDS) and the test genes (UPF2 and SMG7, respectively). G-600 is the long 3'UTR-based NMD reporter, G-95I is the intron-based NMD reporter and G-95 is a non-target negative control construct.
The NMD reporters and the control are shown as transcripts. conducted. Note that the same RT-PCR assay is also shown at Figure 5C.   (11) and 17 dicot Barentsz mRNA have long 3'UTR with NMD relevant introns,while 10 dicot Barentsz transcripts have exclusively intron-based NMD sensitive 3'UTR. 4/42 have NMD insensitive 3'UTR and none of them is exclusively sensitive to the long 3'UTR-based NMD.