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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 nonsense codon effect on the β-globin pre-mRNA abundance exhibits cell line specificity.(A) HeLa cells were stably transfected with the βWT or β39 constructs as indicated below the histogram. Total RNA was isolated and βWT and β39 steady-state mRNA levels were quantified by RT-qPCR using specific primers for the human β-globin processed mRNA, as in Figure 5C, D. The histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (B) Total RNA was also analysed by reverse transcription-coupled quantitative PCR (RT-qPCR), with specific primers for the human β-globin pre-mRNA, as in Figure 5A, B. For each case, intron 1 and intron 2 containing human β-globin pre-RNA levels were determined by normalization to the level of the puromycin resistance mRNA, using the comparative Ct method, and compared to the wild-type control. The percentage pre-mRNA values were plotted for each construct and the histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed as in (A).
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pone-0038505-g007: The nonsense codon effect on the β-globin pre-mRNA abundance exhibits cell line specificity.(A) HeLa cells were stably transfected with the βWT or β39 constructs as indicated below the histogram. Total RNA was isolated and βWT and β39 steady-state mRNA levels were quantified by RT-qPCR using specific primers for the human β-globin processed mRNA, as in Figure 5C, D. The histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (B) Total RNA was also analysed by reverse transcription-coupled quantitative PCR (RT-qPCR), with specific primers for the human β-globin pre-mRNA, as in Figure 5A, B. For each case, intron 1 and intron 2 containing human β-globin pre-RNA levels were determined by normalization to the level of the puromycin resistance mRNA, using the comparative Ct method, and compared to the wild-type control. The percentage pre-mRNA values were plotted for each construct and the histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed as in (A).

Mentions: To assess whether the reduced nonsense pre-mRNA levels phenotype is cell line-specific, we next analyzed the abundance of βWT and β39 pre-mRNAs in non-erythroid cells. Thus, HeLa cells were stably transfected with the βWT or β39 genes, which were previously cloned into the pTRE2 vector, behind the human cytomegalovirus promoter. The corresponding stably expressed spliced and unspliced human β-globin transcript levels were quantified by RT-qPCR analyses as before (Figure 7). Although the PTC-bearing β-globin mRNA steady-state level is downregulated (Figure 7A), as expected for a transcript typically committed to NMD [32]–[35], the corresponding β39 unspliced RNA steady-state level is neither lower nor significantly different relatively to the βWT control (P>0.05) (Figure 7B).


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 nonsense codon effect on the β-globin pre-mRNA abundance exhibits cell line specificity.(A) HeLa cells were stably transfected with the βWT or β39 constructs as indicated below the histogram. Total RNA was isolated and βWT and β39 steady-state mRNA levels were quantified by RT-qPCR using specific primers for the human β-globin processed mRNA, as in Figure 5C, D. The histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (B) Total RNA was also analysed by reverse transcription-coupled quantitative PCR (RT-qPCR), with specific primers for the human β-globin pre-mRNA, as in Figure 5A, B. For each case, intron 1 and intron 2 containing human β-globin pre-RNA levels were determined by normalization to the level of the puromycin resistance mRNA, using the comparative Ct method, and compared to the wild-type control. The percentage pre-mRNA values were plotted for each construct and the histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed as in (A).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366927&req=5

pone-0038505-g007: The nonsense codon effect on the β-globin pre-mRNA abundance exhibits cell line specificity.(A) HeLa cells were stably transfected with the βWT or β39 constructs as indicated below the histogram. Total RNA was isolated and βWT and β39 steady-state mRNA levels were quantified by RT-qPCR using specific primers for the human β-globin processed mRNA, as in Figure 5C, D. The histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (B) Total RNA was also analysed by reverse transcription-coupled quantitative PCR (RT-qPCR), with specific primers for the human β-globin pre-mRNA, as in Figure 5A, B. For each case, intron 1 and intron 2 containing human β-globin pre-RNA levels were determined by normalization to the level of the puromycin resistance mRNA, using the comparative Ct method, and compared to the wild-type control. The percentage pre-mRNA values were plotted for each construct and the histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed as in (A).
Mentions: To assess whether the reduced nonsense pre-mRNA levels phenotype is cell line-specific, we next analyzed the abundance of βWT and β39 pre-mRNAs in non-erythroid cells. Thus, HeLa cells were stably transfected with the βWT or β39 genes, which were previously cloned into the pTRE2 vector, behind the human cytomegalovirus promoter. The corresponding stably expressed spliced and unspliced human β-globin transcript levels were quantified by RT-qPCR analyses as before (Figure 7). Although the PTC-bearing β-globin mRNA steady-state level is downregulated (Figure 7A), as expected for a transcript typically committed to NMD [32]–[35], the corresponding β39 unspliced RNA steady-state level is neither lower nor significantly different relatively to the βWT control (P>0.05) (Figure 7B).

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