Inter-kingdom conservation of mechanism of nonsense-mediated mRNA decay

Abstract

Nonsense-mediated mRNA decay (NMD) is a quality control system that degrades mRNAs containing premature termination codons. Although NMD is well characterized in yeast and mammals, plant NMD is poorly understood. We have undertaken the functional dissection of NMD pathways in plants. Using an approach that allows rapid identification of plant NMD trans factors, we demonstrated that two plant NMD pathways coexist, one eliminates mRNAs with long 3'UTRs, whereas a distinct pathway degrades mRNAs harbouring 3'UTR-located introns. We showed that UPF1, UPF2 and SMG-7 are involved in both plant NMD pathways, whereas Mago and Y14 are required only for intron-based NMD. The molecular mechanism of long 3'UTR-based plant NMD resembled yeast NMD, whereas the intron-based NMD was similar to mammalian NMD, suggesting that both pathways are evolutionarily conserved. Interestingly, the SMG-7 NMD component is targeted by NMD, suggesting that plant NMD is autoregulated. We propose that a complex, autoregulated NMD mechanism operated in stem eukaryotes, and that despite aspect of the mechanism being simplified in different lineages, feedback regulation was retained in all kingdoms.

Figure 1

Roles of UPFs in plant NMD. (A) Schematic representation of NMD constructs. G-L contains a 600 nt stuffer sequence cloned between the stop and the 35S terminator (35sT). Gc contains a 200 nt stuffer region. Gc-I is the same as Gc but contains an intron (I) between the c-stuffer and the terminator. (B) VIGS-NMD system. N. benthamiana plants are infected with TRV-P or a modified TRV-P harbouring a sequence from a candidate NMD trans factor (TRV-P-candidate). NMD activity of the silenced leaves is tested by agroinfiltration with control (con) and NMD test constructs. (C–E) Long 3′UTR-based NMD is inhibited in UPF-silenced plants. Leaves of P-silenced control (P) or U1-, U2- and U3-silenced test plants (U1, U2, U3) were infiltrated with G-L NMD test constructs (test constructs are shown as bold letters) or with GFP (as a control to show that agroinfiltration worked well in silenced plants). P14 was co-infiltrated with each construct. RNAs isolated at 3 d.p.i. were analysed in gel blot assays using P14 and GFP probes. GFP or G-L transcript levels were normalized to the corresponding P14 mRNA levels. Mean values were calculated from three independent experiments, and then these mean transcript levels were compared and graphically presented. (D, E) Mean values of GFP (D) or G-L mRNA levels (E) of P control leaves are taken as 1 and the corresponding transcript levels of U1-, U2- and U3-silenced leaves are shown relative to it. At NMD test constructs, numbers >1 indicate that the NMD is inhibited. s.d. is indicated by error bar. (F–H) Intron-based NMD activity in UPF-silenced plants. Leaves of silenced plants were infiltrated with Gc control or with Gc-I (bold letters) intron-based NMD test constructs. As processed Gc and Gc-I mRNAs are identical, they run to the same position on RNA gel blot. Gc and Gc-I mRNA levels were normalized and the mean values were calculated as described above. (G, H) Graphical representation of Gc or Gc-I expression in UPF-silenced plants relative to P-silenced control. Note that Gc and Gc-I expressions are similarly increased in U3-silenced leaves relative to P-silenced control, whereas in U1- or U2-silenced leaves Gc-I transcript levels are much more increased than Gc levels.

Figure 2

UPF1, UPF2, UPF3, SMG-7 and Y14 are required for plant NMD. (A) Leaves of silenced control (P) and U1-, U2-, U3-, SMG-7- and Y14-silenced test plants (U1, U2, U3, SMG-7 and Y14) were infiltrated with GFP and Gc control constructs or were infiltrated with G-L or Gc-I NMD test constructs. UV pictures were taken at 3 d.p.i. Fluorescence of the infiltrated silenced plant should be compared with the corresponding patch on P control. Enhanced fluorescence indicates that the silenced gene is required for that type of NMD. (B) Complementation of SMG-7-silenced plants. Leaves of SMG-7-silenced plants were infiltrated with Gc-I or were co-infiltrated with Gc-I and with FLAG-tagged Arabidopsis SMG-7 (SMG-7). Complementation restores NMD activity leading to reduced Gc-I accumulation, which manifests in lowered fluorescence. (C) Y14 complementation. Y14-silenced leaves were infiltrated with Gc-I or were co-infiltrated with Gc-I and with either Arabidopsis (AtY14) or N. benthamiana Y14 (NbY14). Note that only NbY14 complemented the NMD deficiency of Y14-silenced leaf. (D) Position of poly(A) tail has a role in PTC definition. G-L control and PABP localization test constructs (G-203A-L, G-81A-L) were infiltrated or co-infiltrated with a dominant-negative UPF1 (U1DN) construct into N. benthamiana leaves.

Figure 3

SMG-7 is required for both types of NMD. (A–C) Effect of SMG-7 silencing on long 3′UTR-based NMD. Silenced control (P) and SMG-7-silenced test plants (SMG-7) were infiltrated with GFP control and G-L NMD test constructs. (D–F) The effect of SMG-7 silencing on intron-based NMD. P- and SMG-7-silenced plants were infiltrated with Gc control or Gc-I NMD test constructs. Panels B, C, E and F are the graphical representations of these experiments. Expression of each construct in SMG-7-silenced plant is shown relative to P-silenced control.

Figure 4

Role of Mago and Y14 in plant NMD. (A–F) Mago and Y14 are not involved in long 3′UTR-based NMD. Leaves of P-silenced control (P) and Mago- or Y14-silenced test plants (Mago, Y14) were infiltrated with GFP or G-L long 3′UTR-based NMD test construct. (G–L) Effect of Mago and Y14 VIGS on intron-based NMD. Leaves of silenced plants were infiltrated with Gc control or Gc-I NMD test constructs. Gc expression is not enhanced in Mago- or Y14-silenced leaves (H and K). Gc-I expression is enhanced in both Mago- and Y14-silenced plants relative to the P-silenced control (I and L, compare column B with A). To carry out complementation assay, N. benthamiana Mago or Y14 was co-infiltrated with Gc-I test constructs into Mago- and Y14-silenced leaves (G and J, lanes 6). Complementation led to enhanced NMD activity, which manifested in reduced Gc-I expression (G, J, compare lanes 6 with 5; I, L, compare columns D with B).

Figure 5

Overexpression of a mutant Mago or Y14 inhibits intron-based NMD. (A) Interactions of N. benthamiana Mago and Y14. HA-tagged Y14 (Y14–HA) or a Y14 mutant (Y14DN–HA) was co-infiltrated with a FLAG-tagged Mago (Mago–FLAG) or a mutant Mago (MagoDN–FLAG). P14 was co-infiltrated with each sample. At 3 d.p.i., proteins were extracted and HA co-IP was carried out. Input (I) and elutes of precipitate (E) were analysed by western blotting. P14 probe was used as negative controls. E is 20 × concentrated relative to the input, thus equal signal strength at I and E means that IP efficiency was ∼5%. (B–I) Overexpression of Y14 and Mago dominant-negative mutants inhibits intron-based NMD but does not affect long 3′UTR-based NMD. N. benthamiana leaves were infiltrated with G-L (B–E) or Gc-I (F–I) NMD test construct (−) or were co-infiltrated with a dominant-negative UPF1 (U1DN), a wild-type Y14 or a Mago, and a mutant Y14 (Y14DN) or a Mago (MagoDN). Note that the wild-type Y14 (but not Mago) showed mild dominant-negative effect on intron-based NMD, whereas both Y14DN and MagoDN showed strong dominant-negative effect on intron-based NMD.

Figure 6

Role of PABP position in plant PTC definition. (A) Constructs used for artificial PABP localization assays. (B, C) G-L control and test (G-203A-L, G-81A-L) constructs were infiltrated or were co-infiltrated with a dominant-negative UPF1 (U1DN) into N. benthamiana leaves. RNA gel blot was hybridized with P14 and GFP probes.

Figure 7

Interactions between UPF proteins. (A–D) Interactions were analysed by HA co-IP assays. N. benthamiana leaves were co-infiltrated with Arabidopsis UPF1, UPF2 and UPF3 in different combinations. P14 was co-infiltrated with each mixture. Elute (E) is 20 × concentrated relative to the input (I).

Figure 8

SMG-7 is downregulated by NMD. (A) The terminator of Arabidopsis SMG-7 gene triggers NMD. Schematic representation of the constructs. C/polyA shows the annotated polyadenylation sites. Introns are shown as thin lines. (B, C) GFP and G-smg7T test construct were infiltrated (lanes 1 and 3) or were co-infiltrated (lanes 2 and 4) with a dominant-negative UPF1 (U1DN) into N. benthamiana leaves. Bold numbers below the blot show the effect of U1DN co-infiltration on G-smg7T expression. (C) Graphical representation of the expression of each construct relative to GFP. (D) The structure of Arabidopsis and rice SMG-7 3′UTRs is conserved. (E) Model of autoregulation of plant NMD.