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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

The mRNP anisotropy model. (A) An intron enhances NMD only when it is close to the PTC. Plot showing the relationship between the distance of the intron from the PTC and NMD enhancement. The numbers are based on the data reported in this paper; from left to right (close to distant): WT3′ivs (Figure 5B, lane 5), PTC140ivs (Figure 2C, lane 4), PTC140ivs-147 (Figure 6D, lane 4), WT-147-3′ivs (Figure 5E, lane 4), PTC27ivs (Figure 2C, lane 3), PTC140-3′ivs (Figure 5B, lane 8), PTC140ivs-291 (Figure 6D, lane 6) and PTC140-5′ivs (Figure 6B, lane 8). The NMD level r corresponds to the fold mRNA reduction relative to the intron-less reporter. (B) Local mRNP anisotropy triggers splice-dependent NMD. Pre-mRNA splicing deposits proteins around either side of the splice junction. These proteins remain associated with the mRNA and change the mRNP local configuration, which in turn affect translation termination or the fate of the post-termination ribosome (e.g. ribosome release). The proteins remain associated with the mRNA only transiently, possibly until the first round of translation. The local mRNP configuration involves interactions of the splice region with the cap-binding complex (CBC) and other proteins associated with the 3′ end. White ovals are nonspecific mRNA-binding proteins; black circles are proteins deposited by splicing. R1, translation release factor 1; R3, translation release factor 3, P, PTC.
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f8: The mRNP anisotropy model. (A) An intron enhances NMD only when it is close to the PTC. Plot showing the relationship between the distance of the intron from the PTC and NMD enhancement. The numbers are based on the data reported in this paper; from left to right (close to distant): WT3′ivs (Figure 5B, lane 5), PTC140ivs (Figure 2C, lane 4), PTC140ivs-147 (Figure 6D, lane 4), WT-147-3′ivs (Figure 5E, lane 4), PTC27ivs (Figure 2C, lane 3), PTC140-3′ivs (Figure 5B, lane 8), PTC140ivs-291 (Figure 6D, lane 6) and PTC140-5′ivs (Figure 6B, lane 8). The NMD level r corresponds to the fold mRNA reduction relative to the intron-less reporter. (B) Local mRNP anisotropy triggers splice-dependent NMD. Pre-mRNA splicing deposits proteins around either side of the splice junction. These proteins remain associated with the mRNA and change the mRNP local configuration, which in turn affect translation termination or the fate of the post-termination ribosome (e.g. ribosome release). The proteins remain associated with the mRNA only transiently, possibly until the first round of translation. The local mRNP configuration involves interactions of the splice region with the cap-binding complex (CBC) and other proteins associated with the 3′ end. White ovals are nonspecific mRNA-binding proteins; black circles are proteins deposited by splicing. R1, translation release factor 1; R3, translation release factor 3, P, PTC.

Mentions: Notably, we found that splicing enhances NMD only when the PTC is not more than 240 bases away from the intron (Figure 8A). Reporters in which we have artificially increased the distance between intron and PTC are no longer subjected to NMD, yet our control experiments indicate that steady-state translation efficiency is not affected. Similarly, lengthening the distance between stop codon and a 3′-UTR intron suppresses NMD but clearly not translation yield. These data indicate that pre-mRNA splicing impinges on NMD directly, yet they exclude that an EJC-like protein complex mediates the effect. Although we still postulate that proteins that remain associated with the mRNA after splicing influence NMD, we propose that the effect is not due to a specific factor deposited at a particular distance before the splice junction (Le Hir et al, 2000, 2001). Instead, we postulate that splicing simply changes the composition or conformation of the mRNP at either side of the junction, and that the local mRNP structure influences translation termination or the release of the post-termination ribosome when the PTC is near but not when it is at a distance from the splice region (Figure 8B). The model predicts that NMD occurs because such perturbations on the ribosome destabilize the mRNP, possibly by preventing establishment of a stable translation circuit as suggested earlier (Brogna and Wen, 2009). It is also possible that there are connections between the spliced region and mRNP components outside the coding region, which could affect the local mRNP; some of the physical interactions of splicing with capping and 3′-end formation, which occur during pre-mRNA processing, may be maintained in the mature mRNP (Proudfoot et al, 2002). In particular, interactions between cap-binding complex (CBC) and splicing have been reported in S. cerevisiae (Colot et al, 1996; Fortes et al, 1999). Notably the yeast CBC can interact with the translation initiation factor eIF4G, and the CBP80 subunit of CBC has structural similarity with eIF4G (Fortes et al, 2000; Marintchev and Wagner, 2005); furthermore, in mammalian cells, NMD substrates are preferentially associated with CBC rather than the translation initiation factor eIF4E (Ishigaki et al, 2001). In summary, we propose that splicing enhances NMD because it influences the structure of the mRNP around the spliced region (the mRNP inisotropy model, Figure 8B). An important assumption of the model is that such anisotropy in mRNP composition and structure is only transient; the prediction is that splicing might only enhance NMD of newly synthesized transcripts before disassociation of the ‘processing' factors. Instead—provided that the PTC is in a region not subjected to splicing-independent NMD—the transcripts are stable afterward. The observation that steady-state mRNA is immune to splicing-dependent NMD in mammalian cells is in agreement with this model (reviewed in Maquat, 2004).


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

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

The mRNP anisotropy model. (A) An intron enhances NMD only when it is close to the PTC. Plot showing the relationship between the distance of the intron from the PTC and NMD enhancement. The numbers are based on the data reported in this paper; from left to right (close to distant): WT3′ivs (Figure 5B, lane 5), PTC140ivs (Figure 2C, lane 4), PTC140ivs-147 (Figure 6D, lane 4), WT-147-3′ivs (Figure 5E, lane 4), PTC27ivs (Figure 2C, lane 3), PTC140-3′ivs (Figure 5B, lane 8), PTC140ivs-291 (Figure 6D, lane 6) and PTC140-5′ivs (Figure 6B, lane 8). The NMD level r corresponds to the fold mRNA reduction relative to the intron-less reporter. (B) Local mRNP anisotropy triggers splice-dependent NMD. Pre-mRNA splicing deposits proteins around either side of the splice junction. These proteins remain associated with the mRNA and change the mRNP local configuration, which in turn affect translation termination or the fate of the post-termination ribosome (e.g. ribosome release). The proteins remain associated with the mRNA only transiently, possibly until the first round of translation. The local mRNP configuration involves interactions of the splice region with the cap-binding complex (CBC) and other proteins associated with the 3′ end. White ovals are nonspecific mRNA-binding proteins; black circles are proteins deposited by splicing. R1, translation release factor 1; R3, translation release factor 3, P, PTC.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: The mRNP anisotropy model. (A) An intron enhances NMD only when it is close to the PTC. Plot showing the relationship between the distance of the intron from the PTC and NMD enhancement. The numbers are based on the data reported in this paper; from left to right (close to distant): WT3′ivs (Figure 5B, lane 5), PTC140ivs (Figure 2C, lane 4), PTC140ivs-147 (Figure 6D, lane 4), WT-147-3′ivs (Figure 5E, lane 4), PTC27ivs (Figure 2C, lane 3), PTC140-3′ivs (Figure 5B, lane 8), PTC140ivs-291 (Figure 6D, lane 6) and PTC140-5′ivs (Figure 6B, lane 8). The NMD level r corresponds to the fold mRNA reduction relative to the intron-less reporter. (B) Local mRNP anisotropy triggers splice-dependent NMD. Pre-mRNA splicing deposits proteins around either side of the splice junction. These proteins remain associated with the mRNA and change the mRNP local configuration, which in turn affect translation termination or the fate of the post-termination ribosome (e.g. ribosome release). The proteins remain associated with the mRNA only transiently, possibly until the first round of translation. The local mRNP configuration involves interactions of the splice region with the cap-binding complex (CBC) and other proteins associated with the 3′ end. White ovals are nonspecific mRNA-binding proteins; black circles are proteins deposited by splicing. R1, translation release factor 1; R3, translation release factor 3, P, PTC.
Mentions: Notably, we found that splicing enhances NMD only when the PTC is not more than 240 bases away from the intron (Figure 8A). Reporters in which we have artificially increased the distance between intron and PTC are no longer subjected to NMD, yet our control experiments indicate that steady-state translation efficiency is not affected. Similarly, lengthening the distance between stop codon and a 3′-UTR intron suppresses NMD but clearly not translation yield. These data indicate that pre-mRNA splicing impinges on NMD directly, yet they exclude that an EJC-like protein complex mediates the effect. Although we still postulate that proteins that remain associated with the mRNA after splicing influence NMD, we propose that the effect is not due to a specific factor deposited at a particular distance before the splice junction (Le Hir et al, 2000, 2001). Instead, we postulate that splicing simply changes the composition or conformation of the mRNP at either side of the junction, and that the local mRNP structure influences translation termination or the release of the post-termination ribosome when the PTC is near but not when it is at a distance from the splice region (Figure 8B). The model predicts that NMD occurs because such perturbations on the ribosome destabilize the mRNP, possibly by preventing establishment of a stable translation circuit as suggested earlier (Brogna and Wen, 2009). It is also possible that there are connections between the spliced region and mRNP components outside the coding region, which could affect the local mRNP; some of the physical interactions of splicing with capping and 3′-end formation, which occur during pre-mRNA processing, may be maintained in the mature mRNP (Proudfoot et al, 2002). In particular, interactions between cap-binding complex (CBC) and splicing have been reported in S. cerevisiae (Colot et al, 1996; Fortes et al, 1999). Notably the yeast CBC can interact with the translation initiation factor eIF4G, and the CBP80 subunit of CBC has structural similarity with eIF4G (Fortes et al, 2000; Marintchev and Wagner, 2005); furthermore, in mammalian cells, NMD substrates are preferentially associated with CBC rather than the translation initiation factor eIF4E (Ishigaki et al, 2001). In summary, we propose that splicing enhances NMD because it influences the structure of the mRNP around the spliced region (the mRNP inisotropy model, Figure 8B). An important assumption of the model is that such anisotropy in mRNP composition and structure is only transient; the prediction is that splicing might only enhance NMD of newly synthesized transcripts before disassociation of the ‘processing' factors. Instead—provided that the PTC is in a region not subjected to splicing-independent NMD—the transcripts are stable afterward. The observation that steady-state mRNA is immune to splicing-dependent NMD in mammalian cells is in agreement with this model (reviewed in Maquat, 2004).

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