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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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UPF1 and UPF2 depletion cause similar degrees of up-modulation of cellular NMD substrates. (a) Immunoblot analysis of protein lysates from HeLa cells transfected with siRNAs against luciferase as a negative control or UPF2 using a UPF2-specific antibody. Serial dilutions corresponding to 100, 50, 20 or 10% (lanes 1–4) of the initial protein amount from luciferase-siRNA transfected cells were loaded to assess the efficiency of the UPF2 siRNA depletion (lane 5). Reprobing with a tubulin-specific antibody was performed as a loading control. (b) Quantitative RT-PCR analysis of SC35 NMD-sensitive variants, TBL2, GADD45B and NAT9 in cells transfected with UPF1 or UPF2 siRNAs. The UPF1 and UPF2 siRNA treatments were controlled by luciferase siRNA and normalised against GAPDH. Mean and SEs were calculated from three independent experiments.
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Figure 4: UPF1 and UPF2 depletion cause similar degrees of up-modulation of cellular NMD substrates. (a) Immunoblot analysis of protein lysates from HeLa cells transfected with siRNAs against luciferase as a negative control or UPF2 using a UPF2-specific antibody. Serial dilutions corresponding to 100, 50, 20 or 10% (lanes 1–4) of the initial protein amount from luciferase-siRNA transfected cells were loaded to assess the efficiency of the UPF2 siRNA depletion (lane 5). Reprobing with a tubulin-specific antibody was performed as a loading control. (b) Quantitative RT-PCR analysis of SC35 NMD-sensitive variants, TBL2, GADD45B and NAT9 in cells transfected with UPF1 or UPF2 siRNAs. The UPF1 and UPF2 siRNA treatments were controlled by luciferase siRNA and normalised against GAPDH. Mean and SEs were calculated from three independent experiments.

Mentions: The role of NMD in directly modulating the abundance of the TBL2, NAT9 and GADD45B transcripts was further analysed by depleting UPF2, which interacts with UPF1 in the NMD pathway (29,30). The efficient depletion of UPF2 to ∼10% was confirmed by immunoblotting (Figure 4a) and, as a functional control, we assessed the abundance of SC35(A) and SC35(B) isoforms (referred to in the subsequent discussion as SC35). The degree of up-modulation in UPF1-depleted and UPF2-depleted cells was not significantly different for all four analysed transcripts (Figure 4b). Taken together, these results indicate that SC35, TBL2, NAT9 and GADD45B are bona fide NMD targets that depend on both UPF1 and UPF2. Analysis of the structure of these transcripts using sequence databases show that SC35 (A and B), TBL2 and GADD45B possess a termination codon located more than 55 bases from the last exon–exon junction, while NAT9 contains an upstream open reading frame (uORF) (Supplementary Figure 1). These structural features are typical for cellular NMD targets (11,31), which may explain the sensitivity of these endogenous mRNAs to cellular NMD activity.Figure 4.


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)

UPF1 and UPF2 depletion cause similar degrees of up-modulation of cellular NMD substrates. (a) Immunoblot analysis of protein lysates from HeLa cells transfected with siRNAs against luciferase as a negative control or UPF2 using a UPF2-specific antibody. Serial dilutions corresponding to 100, 50, 20 or 10% (lanes 1–4) of the initial protein amount from luciferase-siRNA transfected cells were loaded to assess the efficiency of the UPF2 siRNA depletion (lane 5). Reprobing with a tubulin-specific antibody was performed as a loading control. (b) Quantitative RT-PCR analysis of SC35 NMD-sensitive variants, TBL2, GADD45B and NAT9 in cells transfected with UPF1 or UPF2 siRNAs. The UPF1 and UPF2 siRNA treatments were controlled by luciferase siRNA and normalised against GAPDH. Mean and SEs were calculated from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: UPF1 and UPF2 depletion cause similar degrees of up-modulation of cellular NMD substrates. (a) Immunoblot analysis of protein lysates from HeLa cells transfected with siRNAs against luciferase as a negative control or UPF2 using a UPF2-specific antibody. Serial dilutions corresponding to 100, 50, 20 or 10% (lanes 1–4) of the initial protein amount from luciferase-siRNA transfected cells were loaded to assess the efficiency of the UPF2 siRNA depletion (lane 5). Reprobing with a tubulin-specific antibody was performed as a loading control. (b) Quantitative RT-PCR analysis of SC35 NMD-sensitive variants, TBL2, GADD45B and NAT9 in cells transfected with UPF1 or UPF2 siRNAs. The UPF1 and UPF2 siRNA treatments were controlled by luciferase siRNA and normalised against GAPDH. Mean and SEs were calculated from three independent experiments.
Mentions: The role of NMD in directly modulating the abundance of the TBL2, NAT9 and GADD45B transcripts was further analysed by depleting UPF2, which interacts with UPF1 in the NMD pathway (29,30). The efficient depletion of UPF2 to ∼10% was confirmed by immunoblotting (Figure 4a) and, as a functional control, we assessed the abundance of SC35(A) and SC35(B) isoforms (referred to in the subsequent discussion as SC35). The degree of up-modulation in UPF1-depleted and UPF2-depleted cells was not significantly different for all four analysed transcripts (Figure 4b). Taken together, these results indicate that SC35, TBL2, NAT9 and GADD45B are bona fide NMD targets that depend on both UPF1 and UPF2. Analysis of the structure of these transcripts using sequence databases show that SC35 (A and B), TBL2 and GADD45B possess a termination codon located more than 55 bases from the last exon–exon junction, while NAT9 contains an upstream open reading frame (uORF) (Supplementary Figure 1). These structural features are typical for cellular NMD targets (11,31), which may explain the sensitivity of these endogenous mRNAs to cellular NMD activity.Figure 4.

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