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Attenuation of nonsense-mediated mRNA decay facilitates the response to chemotherapeutics.

Popp MW, Maquat LE - Nat Commun (2015)

Bottom Line: Here we show that when human cells are treated with clinically used chemotherapeutic compounds, NMD activity declines partly as a result of the proteolytic production of a dominant-interfering form of the key NMD factor UPF1.Combined exposure of cells to a small-molecule inhibitor of NMD, NMDI-1, and the chemotherapeutic doxorubicin leads to enhanced cell death, while inhibiting UPF1 cleavage protects cells from doxorubicin challenge.We propose a model to explain why the expression levels of genes producing mRNAs of diverse structure that encode proteins of diverse function are under the purview of NMD.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642, USA [2] Center for RNA Biology, University of Rochester, Rochester, New York 14642, USA.

ABSTRACT
Nonsense-mediated mRNA decay (NMD) limits the production of aberrant mRNAs containing a premature termination codon and also controls the levels of endogenous transcripts. Here we show that when human cells are treated with clinically used chemotherapeutic compounds, NMD activity declines partly as a result of the proteolytic production of a dominant-interfering form of the key NMD factor UPF1. Production of cleaved UPF1 functions to upregulate genes involved in the response to apoptotic stresses. The biological consequence is the promotion of cell death. Combined exposure of cells to a small-molecule inhibitor of NMD, NMDI-1, and the chemotherapeutic doxorubicin leads to enhanced cell death, while inhibiting UPF1 cleavage protects cells from doxorubicin challenge. We propose a model to explain why the expression levels of genes producing mRNAs of diverse structure that encode proteins of diverse function are under the purview of NMD.

No MeSH data available.


Related in: MedlinePlus

Characterization of the UPF1 CP mRNP. Western blots of lysates of HEK293T cells transfected with either control (Ctrl) or UPF1 siRNA (100 nM) and 24 h later with plasmid encoding MYC-UPF1-FLAG WT or Δ37-UPF1-FLAG either before (Input) or after anti(α)-FLAG immunoprecipitation (IP), the latter in the presence of BSA (−) or RNase ONE (+). EJC, exon-junction complex constituents. Representative of two independent experiments.
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Figure 6: Characterization of the UPF1 CP mRNP. Western blots of lysates of HEK293T cells transfected with either control (Ctrl) or UPF1 siRNA (100 nM) and 24 h later with plasmid encoding MYC-UPF1-FLAG WT or Δ37-UPF1-FLAG either before (Input) or after anti(α)-FLAG immunoprecipitation (IP), the latter in the presence of BSA (−) or RNase ONE (+). EJC, exon-junction complex constituents. Representative of two independent experiments.

Mentions: We sought to rule out the trivial explanation for the lack of Δ37-UPF1-FLAG function in NMD, i.e. that the truncated protein is misfolded, by characterizing the composition of the RNP containing either MYC-UPF1-FLAG WT or Δ37-UPF1-FLAG. HEK293T cells were depleted of endogenous UPF1 using siRNA, and either MYC-UPF1-FLAG WT or Δ37-UPF1-FLAG was expressed at a level equivalent to the normal level of endogenous UPF1 (Fig. 6). MYC-UPF1-FLAG WT or Δ37-UPF1-FLAG complexes were immunoprecipitated from lysates using anti-FLAG resin, each immunoprecipitate was divided in half, and one half was incubated with BSA while the other half was incubated with RNase ONE to identify protein–protein interactions that are stabilized by RNA.


Attenuation of nonsense-mediated mRNA decay facilitates the response to chemotherapeutics.

Popp MW, Maquat LE - Nat Commun (2015)

Characterization of the UPF1 CP mRNP. Western blots of lysates of HEK293T cells transfected with either control (Ctrl) or UPF1 siRNA (100 nM) and 24 h later with plasmid encoding MYC-UPF1-FLAG WT or Δ37-UPF1-FLAG either before (Input) or after anti(α)-FLAG immunoprecipitation (IP), the latter in the presence of BSA (−) or RNase ONE (+). EJC, exon-junction complex constituents. Representative of two independent experiments.
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Related In: Results  -  Collection

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Figure 6: Characterization of the UPF1 CP mRNP. Western blots of lysates of HEK293T cells transfected with either control (Ctrl) or UPF1 siRNA (100 nM) and 24 h later with plasmid encoding MYC-UPF1-FLAG WT or Δ37-UPF1-FLAG either before (Input) or after anti(α)-FLAG immunoprecipitation (IP), the latter in the presence of BSA (−) or RNase ONE (+). EJC, exon-junction complex constituents. Representative of two independent experiments.
Mentions: We sought to rule out the trivial explanation for the lack of Δ37-UPF1-FLAG function in NMD, i.e. that the truncated protein is misfolded, by characterizing the composition of the RNP containing either MYC-UPF1-FLAG WT or Δ37-UPF1-FLAG. HEK293T cells were depleted of endogenous UPF1 using siRNA, and either MYC-UPF1-FLAG WT or Δ37-UPF1-FLAG was expressed at a level equivalent to the normal level of endogenous UPF1 (Fig. 6). MYC-UPF1-FLAG WT or Δ37-UPF1-FLAG complexes were immunoprecipitated from lysates using anti-FLAG resin, each immunoprecipitate was divided in half, and one half was incubated with BSA while the other half was incubated with RNase ONE to identify protein–protein interactions that are stabilized by RNA.

Bottom Line: Here we show that when human cells are treated with clinically used chemotherapeutic compounds, NMD activity declines partly as a result of the proteolytic production of a dominant-interfering form of the key NMD factor UPF1.Combined exposure of cells to a small-molecule inhibitor of NMD, NMDI-1, and the chemotherapeutic doxorubicin leads to enhanced cell death, while inhibiting UPF1 cleavage protects cells from doxorubicin challenge.We propose a model to explain why the expression levels of genes producing mRNAs of diverse structure that encode proteins of diverse function are under the purview of NMD.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642, USA [2] Center for RNA Biology, University of Rochester, Rochester, New York 14642, USA.

ABSTRACT
Nonsense-mediated mRNA decay (NMD) limits the production of aberrant mRNAs containing a premature termination codon and also controls the levels of endogenous transcripts. Here we show that when human cells are treated with clinically used chemotherapeutic compounds, NMD activity declines partly as a result of the proteolytic production of a dominant-interfering form of the key NMD factor UPF1. Production of cleaved UPF1 functions to upregulate genes involved in the response to apoptotic stresses. The biological consequence is the promotion of cell death. Combined exposure of cells to a small-molecule inhibitor of NMD, NMDI-1, and the chemotherapeutic doxorubicin leads to enhanced cell death, while inhibiting UPF1 cleavage protects cells from doxorubicin challenge. We propose a model to explain why the expression levels of genes producing mRNAs of diverse structure that encode proteins of diverse function are under the purview of NMD.

No MeSH data available.


Related in: MedlinePlus