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. 2006 Nov 6;34(21):6147–6157. doi: 10.1093/nar/gkl737

Figure 4.

Figure 4

Introns located in the 3′-UTR could act as NMD cis elements. (A) Accumulation of the GFP-c and GFP-cLs mRNAs in leaves of N.benthamiana infiltrated with GFP-c + P14 (lanes 1 and 2), GFP-cLs+P14 (lanes 3 and 4), GFP-c + P14 + UPF1DN (lanes 5 and 6) or GFP-cLs + P14 + UPF1DN (lanes 7 and 8) cultures. GFP-c and GFP-cLs mRNA levels were normalized to the corresponding P14 levels, and then mean GFP-c mRNA level was taken as 1. Photos show the GFP activity in the infiltrated leaves. (B) Effect of cycloheximide (Cyc) on the accumulation of the GFP-c and GFP-cLs transcripts. (C) The destabilizing effect of the 3′-UTR located Ls intron is position-dependent. N.tabacum leaves were infiltrated with P-28Ls + PHA + P14 or with P-99Ls + PHA + P14 + UPF1 (lanes 1–2 and 5–6, respectively). These mixtures were also co-infiltrated with UPF1DN (P-28Ls + PHA + P14 + UPF1DN, lanes 9–10, P-99Ls + PHA + P14 + UPF1DN, lanes 13–14). To exclude that the different 3′-UTR length of P-28Ls and P-99Ls transcripts is responsible for the different accumulation, identical but intronless constructs (P-28 and P-99) were infiltrated and analysed in parallel. Blot was hybridized with a PHA-s probe. Upper bands show PHA control transcripts, while the lower bands correspond to P-28Ls, P-28, P-99Ls or P-99 test mRNAs. Test mRNA levels were normalized to the corresponding PHA transcript levels. Note that the OD of the PHA control culture was 0.01, while the ODs of other cultures were 0.2. (D) Effect of cycloheximide (Cyc) on the accumulation of P-28Ls and P-99Ls transcripts. Samples and photos were taken at 3 d.p.i.