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Unspliced precursors of NMD-sensitive β-globin transcripts exhibit decreased steady-state levels in erythroid cells.

Morgado A, Almeida F, Teixeira A, Silva AL, Romão L - PLoS ONE (2012)

Bottom Line: Our analyses by ribonuclease protection assays and reverse transcription-coupled quantitative PCR show that β-globin pre-mRNAs carrying NMD-competent PTCs, but not those containing a NMD-resistant PTC, exhibit a significant decrease in their steady-state levels relatively to the wild-type or to a missense-mutated β-globin pre-mRNA.Functional analyses of these pre-mRNAs in MEL cells demonstrate that their low steady-state levels do not reflect significantly lower pre-mRNA stabilities when compared to the normal control.Furthermore, our results also provide evidence that the relative splicing efficiencies of intron 1 and 2 are unaffected.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética, Instituto Nacional de Saúde Dr Ricardo Jorge, Lisboa, Portugal.

ABSTRACT
Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that detects and rapidly degrades mRNAs carrying premature translation-termination codons (PTCs). Mammalian NMD depends on both splicing and translation, and requires recognition of the premature stop codon by the cytoplasmic ribosomes. Surprisingly, some published data have suggested that nonsense codons may also affect the nuclear metabolism of the nonsense-mutated transcripts. To determine if nonsense codons could influence nuclear events, we have directly assessed the steady-state levels of the unspliced transcripts of wild-type and PTC-containing human β-globin genes stably transfected in mouse erythroleukemia (MEL) cells, after erythroid differentiation induction, or in HeLa cells. Our analyses by ribonuclease protection assays and reverse transcription-coupled quantitative PCR show that β-globin pre-mRNAs carrying NMD-competent PTCs, but not those containing a NMD-resistant PTC, exhibit a significant decrease in their steady-state levels relatively to the wild-type or to a missense-mutated β-globin pre-mRNA. On the contrary, in HeLa cells, human β-globin pre-mRNAs carrying NMD-competent PTCs accumulate at normal levels. Functional analyses of these pre-mRNAs in MEL cells demonstrate that their low steady-state levels do not reflect significantly lower pre-mRNA stabilities when compared to the normal control. Furthermore, our results also provide evidence that the relative splicing efficiencies of intron 1 and 2 are unaffected. This set of data highlights potential nuclear pathways that might be promoter- and/or cell line-specific, which recognize the NMD-sensitive transcripts as abnormal. These specialized nuclear pathway(s) may be superimposed on the general NMD mechanism.

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The half-life of a pre-mRNA carrying an NMD-sensitive PTC is not significantly different from that of the wild-type control pre-mRNA.To determine the pre-mRNA decay kinetics, erythroid differentiated MEL cell pools stably expressing the βWT or the β39 transgenes were incubated with 5 μg/mL of actinomycin D. Total RNA was extracted at the indicated times (0, 15 and 30 min) after actinomycin treatment. Relative pre-mRNA levels were measured by RT-qPCR, as described before. For that, the amount of human β-globin pre-mRNA was normalized against the amount of murine GAPDH mRNA and then re-normalized to the initial time point value (time 0 = 100%). Each point represents the mean and standard error mean from three independent experiments. Linear regression analysis was performed by standard techniques and the difference between slopes was assessed by Student's t test (two-tailed). The half-lives (t1/2) of the pre-mRNAs are indicated.
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pone-0038505-g006: The half-life of a pre-mRNA carrying an NMD-sensitive PTC is not significantly different from that of the wild-type control pre-mRNA.To determine the pre-mRNA decay kinetics, erythroid differentiated MEL cell pools stably expressing the βWT or the β39 transgenes were incubated with 5 μg/mL of actinomycin D. Total RNA was extracted at the indicated times (0, 15 and 30 min) after actinomycin treatment. Relative pre-mRNA levels were measured by RT-qPCR, as described before. For that, the amount of human β-globin pre-mRNA was normalized against the amount of murine GAPDH mRNA and then re-normalized to the initial time point value (time 0 = 100%). Each point represents the mean and standard error mean from three independent experiments. Linear regression analysis was performed by standard techniques and the difference between slopes was assessed by Student's t test (two-tailed). The half-lives (t1/2) of the pre-mRNAs are indicated.

Mentions: As the steady-state level of any unspliced transcript depends on the balance between the rate of its transcription and splicing and/or degradation, we next asked if the low steady-state pre-mRNA levels of the NMD-sensitive transcripts indeed reflect increased decay rates rather than changes at the transcriptional level. Thus, we determined the decay kinetics of the β39 pre-mRNA relatively to that of the wild-type control pre-mRNA stably expressed in MEL cells. For this purpose, we treated the erythroid differentiated βWT and β39 MEL cell pools with actinomycin D to inhibit RNA synthesis. Total RNA was isolated at three time points after actinomycin D treatment. As before, the amount of unspliced human β-globin transcripts was determined by RT-qPCR (Figure 6). Results show that the βWT pre-mRNA has an average half-life of 32 min. The presence of the PTC at position 39 does not significantly accelerate the decay of the reporter pre-mRNA as it results in a half-life of 28 min (P = 0.50) (Figure 6). Although βWT and β39 pre-mRNAs are not similarly abundant, the β39 turns at similar rates of those of βWT pre-mRNA. These similar values of half-lives are in agreement with a previous study by Lim et al. [39]. Our results suggest that low steady-state pre-mRNA levels of NMD-sensitive transcripts might be due to changes at the transcriptional level.


Unspliced precursors of NMD-sensitive β-globin transcripts exhibit decreased steady-state levels in erythroid cells.

Morgado A, Almeida F, Teixeira A, Silva AL, Romão L - PLoS ONE (2012)

The half-life of a pre-mRNA carrying an NMD-sensitive PTC is not significantly different from that of the wild-type control pre-mRNA.To determine the pre-mRNA decay kinetics, erythroid differentiated MEL cell pools stably expressing the βWT or the β39 transgenes were incubated with 5 μg/mL of actinomycin D. Total RNA was extracted at the indicated times (0, 15 and 30 min) after actinomycin treatment. Relative pre-mRNA levels were measured by RT-qPCR, as described before. For that, the amount of human β-globin pre-mRNA was normalized against the amount of murine GAPDH mRNA and then re-normalized to the initial time point value (time 0 = 100%). Each point represents the mean and standard error mean from three independent experiments. Linear regression analysis was performed by standard techniques and the difference between slopes was assessed by Student's t test (two-tailed). The half-lives (t1/2) of the pre-mRNAs are indicated.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366927&req=5

pone-0038505-g006: The half-life of a pre-mRNA carrying an NMD-sensitive PTC is not significantly different from that of the wild-type control pre-mRNA.To determine the pre-mRNA decay kinetics, erythroid differentiated MEL cell pools stably expressing the βWT or the β39 transgenes were incubated with 5 μg/mL of actinomycin D. Total RNA was extracted at the indicated times (0, 15 and 30 min) after actinomycin treatment. Relative pre-mRNA levels were measured by RT-qPCR, as described before. For that, the amount of human β-globin pre-mRNA was normalized against the amount of murine GAPDH mRNA and then re-normalized to the initial time point value (time 0 = 100%). Each point represents the mean and standard error mean from three independent experiments. Linear regression analysis was performed by standard techniques and the difference between slopes was assessed by Student's t test (two-tailed). The half-lives (t1/2) of the pre-mRNAs are indicated.
Mentions: As the steady-state level of any unspliced transcript depends on the balance between the rate of its transcription and splicing and/or degradation, we next asked if the low steady-state pre-mRNA levels of the NMD-sensitive transcripts indeed reflect increased decay rates rather than changes at the transcriptional level. Thus, we determined the decay kinetics of the β39 pre-mRNA relatively to that of the wild-type control pre-mRNA stably expressed in MEL cells. For this purpose, we treated the erythroid differentiated βWT and β39 MEL cell pools with actinomycin D to inhibit RNA synthesis. Total RNA was isolated at three time points after actinomycin D treatment. As before, the amount of unspliced human β-globin transcripts was determined by RT-qPCR (Figure 6). Results show that the βWT pre-mRNA has an average half-life of 32 min. The presence of the PTC at position 39 does not significantly accelerate the decay of the reporter pre-mRNA as it results in a half-life of 28 min (P = 0.50) (Figure 6). Although βWT and β39 pre-mRNAs are not similarly abundant, the β39 turns at similar rates of those of βWT pre-mRNA. These similar values of half-lives are in agreement with a previous study by Lim et al. [39]. Our results suggest that low steady-state pre-mRNA levels of NMD-sensitive transcripts might be due to changes at the transcriptional level.

Bottom Line: Our analyses by ribonuclease protection assays and reverse transcription-coupled quantitative PCR show that β-globin pre-mRNAs carrying NMD-competent PTCs, but not those containing a NMD-resistant PTC, exhibit a significant decrease in their steady-state levels relatively to the wild-type or to a missense-mutated β-globin pre-mRNA.Functional analyses of these pre-mRNAs in MEL cells demonstrate that their low steady-state levels do not reflect significantly lower pre-mRNA stabilities when compared to the normal control.Furthermore, our results also provide evidence that the relative splicing efficiencies of intron 1 and 2 are unaffected.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética, Instituto Nacional de Saúde Dr Ricardo Jorge, Lisboa, Portugal.

ABSTRACT
Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that detects and rapidly degrades mRNAs carrying premature translation-termination codons (PTCs). Mammalian NMD depends on both splicing and translation, and requires recognition of the premature stop codon by the cytoplasmic ribosomes. Surprisingly, some published data have suggested that nonsense codons may also affect the nuclear metabolism of the nonsense-mutated transcripts. To determine if nonsense codons could influence nuclear events, we have directly assessed the steady-state levels of the unspliced transcripts of wild-type and PTC-containing human β-globin genes stably transfected in mouse erythroleukemia (MEL) cells, after erythroid differentiation induction, or in HeLa cells. Our analyses by ribonuclease protection assays and reverse transcription-coupled quantitative PCR show that β-globin pre-mRNAs carrying NMD-competent PTCs, but not those containing a NMD-resistant PTC, exhibit a significant decrease in their steady-state levels relatively to the wild-type or to a missense-mutated β-globin pre-mRNA. On the contrary, in HeLa cells, human β-globin pre-mRNAs carrying NMD-competent PTCs accumulate at normal levels. Functional analyses of these pre-mRNAs in MEL cells demonstrate that their low steady-state levels do not reflect significantly lower pre-mRNA stabilities when compared to the normal control. Furthermore, our results also provide evidence that the relative splicing efficiencies of intron 1 and 2 are unaffected. This set of data highlights potential nuclear pathways that might be promoter- and/or cell line-specific, which recognize the NMD-sensitive transcripts as abnormal. These specialized nuclear pathway(s) may be superimposed on the general NMD mechanism.

Show MeSH
Related in: MedlinePlus