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The abundance of RNPS1, a protein component of the exon junction complex, can determine the variability in efficiency of the Nonsense Mediated Decay pathway.

Viegas MH, Gehring NH, Breit S, Hentze MW, Kulozik AE - Nucleic Acids Res. (2007)

Bottom Line: In a HeLa cell model system, NMD efficiency is shown to be remarkably variable and to represent a stable characteristic of different strains.In one of these strains, low NMD efficiency is shown to be functionally related to the reduced abundance of the exon junction component RNPS1.Furthermore, restoration of functional RNPS1 expression, but not of NMD-inactive mutant proteins, also restores efficient NMD in this model.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology, Hematology and Immunology, Children's Hospital, University of Heidelberg, Im Neuenheimer Feld 150, 69120 Heidelberg, Germany.

ABSTRACT
Nonsense-mediated mRNA decay (NMD) is a molecular pathway of mRNA surveillance that ensures rapid degradation of mRNAs containing premature translation termination codons (PTCs) in eukaryotes. NMD has been shown to also regulate normal gene expression and thus emerged as one of the key post-transcriptional mechanisms of gene regulation. Recently, NMD efficiency has been shown to vary between cell types and individuals thus implicating NMD as a modulator of genetic disease severity. We have now specifically analysed the molecular mechanism of variable NMD efficiency and first established an assay system for the quantification of NMD efficiency, which is based on carefully validated cellular NMD target transcripts. In a HeLa cell model system, NMD efficiency is shown to be remarkably variable and to represent a stable characteristic of different strains. In one of these strains, low NMD efficiency is shown to be functionally related to the reduced abundance of the exon junction component RNPS1. Furthermore, restoration of functional RNPS1 expression, but not of NMD-inactive mutant proteins, also restores efficient NMD in this model. We conclude that cellular concentrations of RNPS1 can modify NMD efficiency and propose that cell type specific co-factor availability represents a novel principle that controls NMD.

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Related in: MedlinePlus

Pre-mRNA and mRNA analysis distinguishes direct from indirect NMD targets in UPF1-depleted cells. Quantitative RT-PCR (LightCycler) for 16 UPF1-sensitive transcripts from cells transfected with luciferase (negative control) or UPF1 siRNAs. The abundance of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. The fold up-modulation of pre-mRNAs and mRNAs by UPF1 depletion (mean ± SE) were calculated from 5–7 independent GAPDH normalized and LUC controlled experiments. Potential direct NMD targets (arrows) were defined as those mRNAs with a mean up-modulation >2-fold and with a mean pre-mRNA up-modulation <2-fold.
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Figure 2: Pre-mRNA and mRNA analysis distinguishes direct from indirect NMD targets in UPF1-depleted cells. Quantitative RT-PCR (LightCycler) for 16 UPF1-sensitive transcripts from cells transfected with luciferase (negative control) or UPF1 siRNAs. The abundance of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. The fold up-modulation of pre-mRNAs and mRNAs by UPF1 depletion (mean ± SE) were calculated from 5–7 independent GAPDH normalized and LUC controlled experiments. Potential direct NMD targets (arrows) were defined as those mRNAs with a mean up-modulation >2-fold and with a mean pre-mRNA up-modulation <2-fold.

Mentions: In order to exclude transcripts that are affected by UPF1 depletion in an NMD-independent, non-post-transcriptional fashion, we analysed mRNA and pre-mRNA levels in a subset of 16 transcripts, chosen because of their strong differential expression in the microarray analyses. In several independent experiments performed on UPF1-depleted HeLa cells that showed efficiently inhibited NMD function (see Figure 1), pre-mRNA and mRNA levels for the selected 16 transcripts were quantified by RT-PCR (Figure 2). The microarray data showing up-regulated mRNA abundance in UPF1-depleted cells could be confirmed by RT-PCR for all 16 transcripts. However, only in the case of TBL2 the abundance of the pre-mRNA remained unchanged while the abundance of the mRNA was up-modulated ∼8-fold. In the case of NAT9, these differences were marginal. In all other 14 RNAs, the abundance of the pre-mRNA and the mRNA did not differ significantly, although in two (KCNJ12, SEPW1) the pre-mRNA remained below the threshold of 2-fold up-regulation, whereas the mRNA was up-regulated to a level of >2-fold. These data suggest that most of these mRNAs are likely up-modulated transcriptionally and do not represent bona fide NMD targets. By implication, these data also suggest that a substantial fraction, likely most of the almost 230 transcripts that are up-modulated by UPF1 depletion in our microarray data are indirect NMD targets.Figure 2.


The abundance of RNPS1, a protein component of the exon junction complex, can determine the variability in efficiency of the Nonsense Mediated Decay pathway.

Viegas MH, Gehring NH, Breit S, Hentze MW, Kulozik AE - Nucleic Acids Res. (2007)

Pre-mRNA and mRNA analysis distinguishes direct from indirect NMD targets in UPF1-depleted cells. Quantitative RT-PCR (LightCycler) for 16 UPF1-sensitive transcripts from cells transfected with luciferase (negative control) or UPF1 siRNAs. The abundance of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. The fold up-modulation of pre-mRNAs and mRNAs by UPF1 depletion (mean ± SE) were calculated from 5–7 independent GAPDH normalized and LUC controlled experiments. Potential direct NMD targets (arrows) were defined as those mRNAs with a mean up-modulation >2-fold and with a mean pre-mRNA up-modulation <2-fold.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1935013&req=5

Figure 2: Pre-mRNA and mRNA analysis distinguishes direct from indirect NMD targets in UPF1-depleted cells. Quantitative RT-PCR (LightCycler) for 16 UPF1-sensitive transcripts from cells transfected with luciferase (negative control) or UPF1 siRNAs. The abundance of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization. The fold up-modulation of pre-mRNAs and mRNAs by UPF1 depletion (mean ± SE) were calculated from 5–7 independent GAPDH normalized and LUC controlled experiments. Potential direct NMD targets (arrows) were defined as those mRNAs with a mean up-modulation >2-fold and with a mean pre-mRNA up-modulation <2-fold.
Mentions: In order to exclude transcripts that are affected by UPF1 depletion in an NMD-independent, non-post-transcriptional fashion, we analysed mRNA and pre-mRNA levels in a subset of 16 transcripts, chosen because of their strong differential expression in the microarray analyses. In several independent experiments performed on UPF1-depleted HeLa cells that showed efficiently inhibited NMD function (see Figure 1), pre-mRNA and mRNA levels for the selected 16 transcripts were quantified by RT-PCR (Figure 2). The microarray data showing up-regulated mRNA abundance in UPF1-depleted cells could be confirmed by RT-PCR for all 16 transcripts. However, only in the case of TBL2 the abundance of the pre-mRNA remained unchanged while the abundance of the mRNA was up-modulated ∼8-fold. In the case of NAT9, these differences were marginal. In all other 14 RNAs, the abundance of the pre-mRNA and the mRNA did not differ significantly, although in two (KCNJ12, SEPW1) the pre-mRNA remained below the threshold of 2-fold up-regulation, whereas the mRNA was up-regulated to a level of >2-fold. These data suggest that most of these mRNAs are likely up-modulated transcriptionally and do not represent bona fide NMD targets. By implication, these data also suggest that a substantial fraction, likely most of the almost 230 transcripts that are up-modulated by UPF1 depletion in our microarray data are indirect NMD targets.Figure 2.

Bottom Line: In a HeLa cell model system, NMD efficiency is shown to be remarkably variable and to represent a stable characteristic of different strains.In one of these strains, low NMD efficiency is shown to be functionally related to the reduced abundance of the exon junction component RNPS1.Furthermore, restoration of functional RNPS1 expression, but not of NMD-inactive mutant proteins, also restores efficient NMD in this model.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatric Oncology, Hematology and Immunology, Children's Hospital, University of Heidelberg, Im Neuenheimer Feld 150, 69120 Heidelberg, Germany.

ABSTRACT
Nonsense-mediated mRNA decay (NMD) is a molecular pathway of mRNA surveillance that ensures rapid degradation of mRNAs containing premature translation termination codons (PTCs) in eukaryotes. NMD has been shown to also regulate normal gene expression and thus emerged as one of the key post-transcriptional mechanisms of gene regulation. Recently, NMD efficiency has been shown to vary between cell types and individuals thus implicating NMD as a modulator of genetic disease severity. We have now specifically analysed the molecular mechanism of variable NMD efficiency and first established an assay system for the quantification of NMD efficiency, which is based on carefully validated cellular NMD target transcripts. In a HeLa cell model system, NMD efficiency is shown to be remarkably variable and to represent a stable characteristic of different strains. In one of these strains, low NMD efficiency is shown to be functionally related to the reduced abundance of the exon junction component RNPS1. Furthermore, restoration of functional RNPS1 expression, but not of NMD-inactive mutant proteins, also restores efficient NMD in this model. We conclude that cellular concentrations of RNPS1 can modify NMD efficiency and propose that cell type specific co-factor availability represents a novel principle that controls NMD.

Show MeSH
Related in: MedlinePlus