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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

Inhibiting UPF1 CP generation protects cells from doxorubicin challenge, and inhibiting NMD promotes doxorubicin-induced cell death. (a) Western blots of HeLa cells stably expressing empty vector (ϴ) or equivalent amounts of MYC-UPF1-FLAG WT or MYC-UPF1-FLAG D37N. Blots derive from (and are representative of) four biological replicate samples used in b. (b) Cell lines from a were plated in 96-well opaque tissue-culture dishes (5,000 cells/well) and exposed to the indicated concentrations of doxorubicin for 16 h. Viable cells were quantitated using a Cell-Titer Glo assay. Data are normalized to untreated cells for each cell line. Errors bar=S.E.M., asterisk=p<0.05 relative to MYC-UPF1-FLAG WT cell-line samples using the Student’s t-test. n=4 biological replicates. (c) Structure of NMDI-1. (d) HeLa cells were plated as in b and exposed to one of three treatments 24 h later: the indicated doxorubicin concentration is provided for 16 h either alone (grey histograms) or in the presence of 10 μM NMDI-1 (blue histograms). Alternatively, cells were pre-incubated with 10 μM NMDI-1 for 8 h, washed three times, and incubated with the indicated concentrations of doxorubicin for 16 h in the absence of NMDI-1. Viable cells were quantitated as in b. Error bars= S.E.M., asterisk p<0.05 relative to doxorubicin alone treatment using the Student’s t-test. n=4 biological replicates. (e) Model implicating NMD modulation in establishing different cellular states (compare white box to the left and gray box to the right) by sculpting the mRNA milieu. Transcription produces mRNAs that are (red) or are not (blue) NMD targets (i.e. are unregulated), or are indirect NMD targets (black). Normally, NMD is active, eliminating direct and indirect NMD targets from the mRNA milieu (left white box). However, NMD activity can be modulated by various perturbations. We show here that production of UPF1 CP(s) at sub-stoichiometric levels downregulates NMD activity, causing direct NMD targets to enter the mRNA milieu, which secondarily causes upregulation of indirect NMD targets (right gray box). Together, these changes sculpt the pool of mRNAs to one that is competent to rapidly respond to the stimulus that elicited the inhibition of NMD. Here, we have shown that the stimulus (doxorubicin) attenuates NMD, facilitating an appropriate response (cell death).
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Figure 8: Inhibiting UPF1 CP generation protects cells from doxorubicin challenge, and inhibiting NMD promotes doxorubicin-induced cell death. (a) Western blots of HeLa cells stably expressing empty vector (ϴ) or equivalent amounts of MYC-UPF1-FLAG WT or MYC-UPF1-FLAG D37N. Blots derive from (and are representative of) four biological replicate samples used in b. (b) Cell lines from a were plated in 96-well opaque tissue-culture dishes (5,000 cells/well) and exposed to the indicated concentrations of doxorubicin for 16 h. Viable cells were quantitated using a Cell-Titer Glo assay. Data are normalized to untreated cells for each cell line. Errors bar=S.E.M., asterisk=p<0.05 relative to MYC-UPF1-FLAG WT cell-line samples using the Student’s t-test. n=4 biological replicates. (c) Structure of NMDI-1. (d) HeLa cells were plated as in b and exposed to one of three treatments 24 h later: the indicated doxorubicin concentration is provided for 16 h either alone (grey histograms) or in the presence of 10 μM NMDI-1 (blue histograms). Alternatively, cells were pre-incubated with 10 μM NMDI-1 for 8 h, washed three times, and incubated with the indicated concentrations of doxorubicin for 16 h in the absence of NMDI-1. Viable cells were quantitated as in b. Error bars= S.E.M., asterisk p<0.05 relative to doxorubicin alone treatment using the Student’s t-test. n=4 biological replicates. (e) Model implicating NMD modulation in establishing different cellular states (compare white box to the left and gray box to the right) by sculpting the mRNA milieu. Transcription produces mRNAs that are (red) or are not (blue) NMD targets (i.e. are unregulated), or are indirect NMD targets (black). Normally, NMD is active, eliminating direct and indirect NMD targets from the mRNA milieu (left white box). However, NMD activity can be modulated by various perturbations. We show here that production of UPF1 CP(s) at sub-stoichiometric levels downregulates NMD activity, causing direct NMD targets to enter the mRNA milieu, which secondarily causes upregulation of indirect NMD targets (right gray box). Together, these changes sculpt the pool of mRNAs to one that is competent to rapidly respond to the stimulus that elicited the inhibition of NMD. Here, we have shown that the stimulus (doxorubicin) attenuates NMD, facilitating an appropriate response (cell death).

Mentions: To test the first hypothesis, we utilized HeLa cell lines stably expressing MYC-UPF1-FLAG WT or non-cleavable MYC-UPF1-FLAG D37N (Figs. 3d, 4b, 8a), both of which support NMD. Each was expressed at ~2.7 fold above the level of endogenous UPF1 and, more importantly, at levels identical to one another (Fig. 8a). We exposed these cell lines to a range of doxorubicin concentrations and assessed cell viability after 16 h using an assay that detects ATP generation by living cells. At a sub-lethal doxorubicin concentration (0.5 μM), no statistically significant difference in viability was detected. However, as doxorubicin toxicity increased, the MYC-UPF1-FLAG D37N cell line showed increased resistance to death relative to the MYC-UPF1-FLAG WT cell line, reaching a maximum of ~2.2-fold greater survival.


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

Popp MW, Maquat LE - Nat Commun (2015)

Inhibiting UPF1 CP generation protects cells from doxorubicin challenge, and inhibiting NMD promotes doxorubicin-induced cell death. (a) Western blots of HeLa cells stably expressing empty vector (ϴ) or equivalent amounts of MYC-UPF1-FLAG WT or MYC-UPF1-FLAG D37N. Blots derive from (and are representative of) four biological replicate samples used in b. (b) Cell lines from a were plated in 96-well opaque tissue-culture dishes (5,000 cells/well) and exposed to the indicated concentrations of doxorubicin for 16 h. Viable cells were quantitated using a Cell-Titer Glo assay. Data are normalized to untreated cells for each cell line. Errors bar=S.E.M., asterisk=p<0.05 relative to MYC-UPF1-FLAG WT cell-line samples using the Student’s t-test. n=4 biological replicates. (c) Structure of NMDI-1. (d) HeLa cells were plated as in b and exposed to one of three treatments 24 h later: the indicated doxorubicin concentration is provided for 16 h either alone (grey histograms) or in the presence of 10 μM NMDI-1 (blue histograms). Alternatively, cells were pre-incubated with 10 μM NMDI-1 for 8 h, washed three times, and incubated with the indicated concentrations of doxorubicin for 16 h in the absence of NMDI-1. Viable cells were quantitated as in b. Error bars= S.E.M., asterisk p<0.05 relative to doxorubicin alone treatment using the Student’s t-test. n=4 biological replicates. (e) Model implicating NMD modulation in establishing different cellular states (compare white box to the left and gray box to the right) by sculpting the mRNA milieu. Transcription produces mRNAs that are (red) or are not (blue) NMD targets (i.e. are unregulated), or are indirect NMD targets (black). Normally, NMD is active, eliminating direct and indirect NMD targets from the mRNA milieu (left white box). However, NMD activity can be modulated by various perturbations. We show here that production of UPF1 CP(s) at sub-stoichiometric levels downregulates NMD activity, causing direct NMD targets to enter the mRNA milieu, which secondarily causes upregulation of indirect NMD targets (right gray box). Together, these changes sculpt the pool of mRNAs to one that is competent to rapidly respond to the stimulus that elicited the inhibition of NMD. Here, we have shown that the stimulus (doxorubicin) attenuates NMD, facilitating an appropriate response (cell death).
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Figure 8: Inhibiting UPF1 CP generation protects cells from doxorubicin challenge, and inhibiting NMD promotes doxorubicin-induced cell death. (a) Western blots of HeLa cells stably expressing empty vector (ϴ) or equivalent amounts of MYC-UPF1-FLAG WT or MYC-UPF1-FLAG D37N. Blots derive from (and are representative of) four biological replicate samples used in b. (b) Cell lines from a were plated in 96-well opaque tissue-culture dishes (5,000 cells/well) and exposed to the indicated concentrations of doxorubicin for 16 h. Viable cells were quantitated using a Cell-Titer Glo assay. Data are normalized to untreated cells for each cell line. Errors bar=S.E.M., asterisk=p<0.05 relative to MYC-UPF1-FLAG WT cell-line samples using the Student’s t-test. n=4 biological replicates. (c) Structure of NMDI-1. (d) HeLa cells were plated as in b and exposed to one of three treatments 24 h later: the indicated doxorubicin concentration is provided for 16 h either alone (grey histograms) or in the presence of 10 μM NMDI-1 (blue histograms). Alternatively, cells were pre-incubated with 10 μM NMDI-1 for 8 h, washed three times, and incubated with the indicated concentrations of doxorubicin for 16 h in the absence of NMDI-1. Viable cells were quantitated as in b. Error bars= S.E.M., asterisk p<0.05 relative to doxorubicin alone treatment using the Student’s t-test. n=4 biological replicates. (e) Model implicating NMD modulation in establishing different cellular states (compare white box to the left and gray box to the right) by sculpting the mRNA milieu. Transcription produces mRNAs that are (red) or are not (blue) NMD targets (i.e. are unregulated), or are indirect NMD targets (black). Normally, NMD is active, eliminating direct and indirect NMD targets from the mRNA milieu (left white box). However, NMD activity can be modulated by various perturbations. We show here that production of UPF1 CP(s) at sub-stoichiometric levels downregulates NMD activity, causing direct NMD targets to enter the mRNA milieu, which secondarily causes upregulation of indirect NMD targets (right gray box). Together, these changes sculpt the pool of mRNAs to one that is competent to rapidly respond to the stimulus that elicited the inhibition of NMD. Here, we have shown that the stimulus (doxorubicin) attenuates NMD, facilitating an appropriate response (cell death).
Mentions: To test the first hypothesis, we utilized HeLa cell lines stably expressing MYC-UPF1-FLAG WT or non-cleavable MYC-UPF1-FLAG D37N (Figs. 3d, 4b, 8a), both of which support NMD. Each was expressed at ~2.7 fold above the level of endogenous UPF1 and, more importantly, at levels identical to one another (Fig. 8a). We exposed these cell lines to a range of doxorubicin concentrations and assessed cell viability after 16 h using an assay that detects ATP generation by living cells. At a sub-lethal doxorubicin concentration (0.5 μM), no statistically significant difference in viability was detected. However, as doxorubicin toxicity increased, the MYC-UPF1-FLAG D37N cell line showed increased resistance to death relative to the MYC-UPF1-FLAG WT cell line, reaching a maximum of ~2.2-fold greater survival.

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