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Splicing-dependent NMD does not require the EJC in Schizosaccharomyces pombe.

Wen J, Brogna S - EMBO J. (2010)

Bottom Line: The exon junction complex (EJC) is believed to mediate the link between splicing and NMD in these systems.Still the effect of splicing seems to be direct-we have found that the important NMD determinant is the proximity of an intron to the PTC, not just the occurrence of splicing.On the basis of these results, we propose a new model to explain how splicing could affect NMD.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.

ABSTRACT
Nonsense-mediated mRNA decay (NMD) is a translation-linked process that destroys mRNAs with premature translation termination codons (PTCs). In mammalian cells, NMD is also linked to pre-mRNA splicing, usually PTCs trigger strong NMD only when positioned upstream of at least one intron. The exon junction complex (EJC) is believed to mediate the link between splicing and NMD in these systems. Here, we report that in Schizosaccharomyces pombe splicing also enhances NMD, but against the EJC model prediction, an intron stimulated NMD regardless of whether it is positioned upstream or downstream of the PTC and EJC components are not required. Still the effect of splicing seems to be direct-we have found that the important NMD determinant is the proximity of an intron to the PTC, not just the occurrence of splicing. On the basis of these results, we propose a new model to explain how splicing could affect NMD.

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Related in: MedlinePlus

Lengthening the 3′ UTR and lack of PABPC only modestly affect NMD. (A) Diagram of reporters with different length 3′ UTRs. The WT-102 reporter carries a 102 bp insert 9 bp after the normal stop codon, WT-418, a 418 bp at the same position. WT-402 contains a 402 bp insert 6 bp upstream of the normal stop codon, in frame with the GFP ORF. (B–F) Northern blot analysis of total RNA from wild-type (B, C, E) and upf2Δ (D, F) cells transformed with the indicated reporters. The top panels show hybridizations with the GFP probe, bottom panels with the Rpl32 probe. Values are percentages with s.d. based on three independent experiments. (G) Northern blot analysis of total RNA from pab1Δ cells transformed with the indicated reporters. The top panel shows hybridization with the GFP probe, bottom panel with the Rpl32 probe. Quantifications of the GFP mRNA are as above, based on two independent experiments.
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f7: Lengthening the 3′ UTR and lack of PABPC only modestly affect NMD. (A) Diagram of reporters with different length 3′ UTRs. The WT-102 reporter carries a 102 bp insert 9 bp after the normal stop codon, WT-418, a 418 bp at the same position. WT-402 contains a 402 bp insert 6 bp upstream of the normal stop codon, in frame with the GFP ORF. (B–F) Northern blot analysis of total RNA from wild-type (B, C, E) and upf2Δ (D, F) cells transformed with the indicated reporters. The top panels show hybridizations with the GFP probe, bottom panels with the Rpl32 probe. Values are percentages with s.d. based on three independent experiments. (G) Northern blot analysis of total RNA from pab1Δ cells transformed with the indicated reporters. The top panel shows hybridization with the GFP probe, bottom panel with the Rpl32 probe. Quantifications of the GFP mRNA are as above, based on two independent experiments.

Mentions: The faux 3′-UTR model predicts that NMD should increase with the distance of the PTC from the 3′ end. Our observation that NMD is less apparent when the PTC is located in the second half of the gene seems to be, at least in intron-less reporters, consistent with the model. To test it directly, first we inserted either a 102 or 418 bp fragment just downstream of the stop codon (Figure 7A). The expectation was that both insertions would enhance NMD because termination occurs further away from the 3′ end. Lengthening the 3′ UTR by 102 bases should make PTC27 behave like PTC6, enhancing NMD, whereas lengthening the 3′ UTR by 418 bases would trigger NMD of the PTC140 transcript; the insertion places the mutation at the same distance from the 3′ end as PTC6 in the original reporter. We found that the 102-base insertion reproducibly enhanced NMD of the mRNA with PTC27 (Figure 7B, lane 3 versus 7), but did not affect that with PTC6 (lane 2 versus 6). Surprisingly, the level of the PTC140 mRNA was constantly higher than that with the original 3′ UTR rather than being reduced (Figure 7B, lane 4 versus 8). In the reporters with the 418-base insertion, we found that the mRNA without PTCs was reduced to about 63% of that with the normal 3′ UTR (Figure 7C); the mRNA level was restored in the upf2Δ strain (Figure 7D), consistent with the view that mRNAs with artificially long 3′ UTR become NMD substrates. However, we found no linear relationship between mRNA levels and distance of the PTC from the 3′ end. For example, it was expected that the 418-bp insertion would make PTC140 behave like PTC6 in the initial reporter, triggering strong NMD. Instead, we found that the PTC140 mRNA level was only half of that of the reporter with the original 3′ UTR (Figure 7C, lane 1 versus 5), and it was barely reduced relative to the PTC-less control (Figure 7C, lane 2 versus 5). To further assess to what extent the distance from the 3′ end determines NMD, we inserted an in-frame 402 bp fragment just before the normal stop codon (Figure 7A)—we reasoned that an insertion in the coding region is less likely to influence mRNA stability features that might be present in the original 3′ UTR. We found that PTCs at positions 6 and 27 caused as strong NMD as in the earlier reporters. However, the PTC140 mRNA was only modestly reduced, even though the PTC is at the same distance from the 3′ end as the NMD inducing PTC6 was in the constructs with the original 3′ UTR (Figure 7E and F).


Splicing-dependent NMD does not require the EJC in Schizosaccharomyces pombe.

Wen J, Brogna S - EMBO J. (2010)

Lengthening the 3′ UTR and lack of PABPC only modestly affect NMD. (A) Diagram of reporters with different length 3′ UTRs. The WT-102 reporter carries a 102 bp insert 9 bp after the normal stop codon, WT-418, a 418 bp at the same position. WT-402 contains a 402 bp insert 6 bp upstream of the normal stop codon, in frame with the GFP ORF. (B–F) Northern blot analysis of total RNA from wild-type (B, C, E) and upf2Δ (D, F) cells transformed with the indicated reporters. The top panels show hybridizations with the GFP probe, bottom panels with the Rpl32 probe. Values are percentages with s.d. based on three independent experiments. (G) Northern blot analysis of total RNA from pab1Δ cells transformed with the indicated reporters. The top panel shows hybridization with the GFP probe, bottom panel with the Rpl32 probe. Quantifications of the GFP mRNA are as above, based on two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2876954&req=5

f7: Lengthening the 3′ UTR and lack of PABPC only modestly affect NMD. (A) Diagram of reporters with different length 3′ UTRs. The WT-102 reporter carries a 102 bp insert 9 bp after the normal stop codon, WT-418, a 418 bp at the same position. WT-402 contains a 402 bp insert 6 bp upstream of the normal stop codon, in frame with the GFP ORF. (B–F) Northern blot analysis of total RNA from wild-type (B, C, E) and upf2Δ (D, F) cells transformed with the indicated reporters. The top panels show hybridizations with the GFP probe, bottom panels with the Rpl32 probe. Values are percentages with s.d. based on three independent experiments. (G) Northern blot analysis of total RNA from pab1Δ cells transformed with the indicated reporters. The top panel shows hybridization with the GFP probe, bottom panel with the Rpl32 probe. Quantifications of the GFP mRNA are as above, based on two independent experiments.
Mentions: The faux 3′-UTR model predicts that NMD should increase with the distance of the PTC from the 3′ end. Our observation that NMD is less apparent when the PTC is located in the second half of the gene seems to be, at least in intron-less reporters, consistent with the model. To test it directly, first we inserted either a 102 or 418 bp fragment just downstream of the stop codon (Figure 7A). The expectation was that both insertions would enhance NMD because termination occurs further away from the 3′ end. Lengthening the 3′ UTR by 102 bases should make PTC27 behave like PTC6, enhancing NMD, whereas lengthening the 3′ UTR by 418 bases would trigger NMD of the PTC140 transcript; the insertion places the mutation at the same distance from the 3′ end as PTC6 in the original reporter. We found that the 102-base insertion reproducibly enhanced NMD of the mRNA with PTC27 (Figure 7B, lane 3 versus 7), but did not affect that with PTC6 (lane 2 versus 6). Surprisingly, the level of the PTC140 mRNA was constantly higher than that with the original 3′ UTR rather than being reduced (Figure 7B, lane 4 versus 8). In the reporters with the 418-base insertion, we found that the mRNA without PTCs was reduced to about 63% of that with the normal 3′ UTR (Figure 7C); the mRNA level was restored in the upf2Δ strain (Figure 7D), consistent with the view that mRNAs with artificially long 3′ UTR become NMD substrates. However, we found no linear relationship between mRNA levels and distance of the PTC from the 3′ end. For example, it was expected that the 418-bp insertion would make PTC140 behave like PTC6 in the initial reporter, triggering strong NMD. Instead, we found that the PTC140 mRNA level was only half of that of the reporter with the original 3′ UTR (Figure 7C, lane 1 versus 5), and it was barely reduced relative to the PTC-less control (Figure 7C, lane 2 versus 5). To further assess to what extent the distance from the 3′ end determines NMD, we inserted an in-frame 402 bp fragment just before the normal stop codon (Figure 7A)—we reasoned that an insertion in the coding region is less likely to influence mRNA stability features that might be present in the original 3′ UTR. We found that PTCs at positions 6 and 27 caused as strong NMD as in the earlier reporters. However, the PTC140 mRNA was only modestly reduced, even though the PTC is at the same distance from the 3′ end as the NMD inducing PTC6 was in the constructs with the original 3′ UTR (Figure 7E and F).

Bottom Line: The exon junction complex (EJC) is believed to mediate the link between splicing and NMD in these systems.Still the effect of splicing seems to be direct-we have found that the important NMD determinant is the proximity of an intron to the PTC, not just the occurrence of splicing.On the basis of these results, we propose a new model to explain how splicing could affect NMD.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.

ABSTRACT
Nonsense-mediated mRNA decay (NMD) is a translation-linked process that destroys mRNAs with premature translation termination codons (PTCs). In mammalian cells, NMD is also linked to pre-mRNA splicing, usually PTCs trigger strong NMD only when positioned upstream of at least one intron. The exon junction complex (EJC) is believed to mediate the link between splicing and NMD in these systems. Here, we report that in Schizosaccharomyces pombe splicing also enhances NMD, but against the EJC model prediction, an intron stimulated NMD regardless of whether it is positioned upstream or downstream of the PTC and EJC components are not required. Still the effect of splicing seems to be direct-we have found that the important NMD determinant is the proximity of an intron to the PTC, not just the occurrence of splicing. On the basis of these results, we propose a new model to explain how splicing could affect NMD.

Show MeSH
Related in: MedlinePlus