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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 presence of the nonsense codon equally decreases the abundance of intron 1 versus intron 2 containing human β-globin pre-mRNAs.(A) Schematic representation of the human β-globin pre-mRNA, as in Figures 3D and 4D. The two pairs of arrows represent the coordinates of both amplicons obtained in the qPCR reactions: intron1-exon2 and exon2-intron2 amplicons. (B) MEL cells were stably transfected with a test human β-globin construct as specified below the histogram. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by reverse transcription-coupled quantitative PCR (RT-qPCR), with specific primers for the human β-globin pre-mRNA, as shown in (A). For each case, intron 1 and intron 2 containing human β-globin pre-RNAs levels were determined by normalization to the level of murine glyceraldehyde 3-phosphate dehydrogenase (GAPDH) 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 using Student's t test (unpaired, two-tailed). (C) Schematic representation of the studied human β-globin mRNAs as in Figures 3A and 4A. The pair of arrows represents the coordinates of the amplicon obtained in the qPCR reactions: exon2–3 amplicon. (D) Human β-globin mRNA quantification was performed by RT-qPCR as in (B), but using specific primers for the human β-globin processed mRNA. Levels of each human β-globin mRNA variant were determined by normalization to the level of murine GAPDH mRNA, using the comparative Ct method, and compared to the wild-type control. The histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed as in (B).
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pone-0038505-g005: The presence of the nonsense codon equally decreases the abundance of intron 1 versus intron 2 containing human β-globin pre-mRNAs.(A) Schematic representation of the human β-globin pre-mRNA, as in Figures 3D and 4D. The two pairs of arrows represent the coordinates of both amplicons obtained in the qPCR reactions: intron1-exon2 and exon2-intron2 amplicons. (B) MEL cells were stably transfected with a test human β-globin construct as specified below the histogram. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by reverse transcription-coupled quantitative PCR (RT-qPCR), with specific primers for the human β-globin pre-mRNA, as shown in (A). For each case, intron 1 and intron 2 containing human β-globin pre-RNAs levels were determined by normalization to the level of murine glyceraldehyde 3-phosphate dehydrogenase (GAPDH) 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 using Student's t test (unpaired, two-tailed). (C) Schematic representation of the studied human β-globin mRNAs as in Figures 3A and 4A. The pair of arrows represents the coordinates of the amplicon obtained in the qPCR reactions: exon2–3 amplicon. (D) Human β-globin mRNA quantification was performed by RT-qPCR as in (B), but using specific primers for the human β-globin processed mRNA. Levels of each human β-globin mRNA variant were determined by normalization to the level of murine GAPDH mRNA, using the comparative Ct method, and compared to the wild-type control. The histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed as in (B).

Mentions: In order to test to what extent the presence of the nonsense codon affects the relative amount of intron 1 versus intron 2 containing β-globin pre-mRNAs, we analysed the β39 and β62 transcripts stably expressed in differentiated MEL cell pools and results were compared to those of the βWT, β127 and β39missense control transcripts. This analysis was carried out by reverse transcription-coupled quantitative PCR (RT-qPCR) assays to specifically quantify the amount of either intron 1 or intron 2 containing human β-globin pre-mRNAs (Figure 5). Thus, pre-mRNA quantification was carried out with two sets of primers specific for the human β-globin intron 1 and intron 2 pre-mRNA sequence, respectively, using a set of primers specific for the murine GAPDH mRNA as an internal control (Figure 5A, B). As a control, RT-qPCR was also performed with a set of specific primers to quantify processed mRNA, to show that, under these experimental conditions, the PTCs at position 39 or 62 are able to induce a strong downregulation of the steady-state levels as expected for mRNAs typically committed to NMD, while levels of mRNA bearing a PTC at the 3′-most exon (β127) are not significantly different from the normal control (Figure 5C, D). The quantitative PCR efficiency for all amplicons was found to be similar and near to 100%. Control reactions using total RNA samples from untransfected MEL cells, confirmed that unspecific amplification of the murine β-globin transcripts was negligible. In agreement with the previously obtained RPA data, RT-qPCR analysis of the intron 2-containing pre-mRNA steady-state levels shows a significant 2.3 to 3.8-fold reduction of the β39 and β62 unspliced RNAs relatively to the βWT pre-mRNA (P<0.01) (Figure 5B). On the other hand, β127 and β39missense unspliced transcripts exhibit similar levels, which are not significantly different from the normal control (P = 0.12 and P = 0.08, respectively). Additionally, in each case, both β-globin intron 1 and intron 2 containing pre-mRNAs yielded very similar expression levels (P>0.05). Therefore, the presence of the NMD-sensitive nonsense codons does not differentially affect the rates of removal of intron 1 and 2, and, thus, splicing efficiency in transcripts bearing NMD-competent nonsense codons seems to be normal.


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 presence of the nonsense codon equally decreases the abundance of intron 1 versus intron 2 containing human β-globin pre-mRNAs.(A) Schematic representation of the human β-globin pre-mRNA, as in Figures 3D and 4D. The two pairs of arrows represent the coordinates of both amplicons obtained in the qPCR reactions: intron1-exon2 and exon2-intron2 amplicons. (B) MEL cells were stably transfected with a test human β-globin construct as specified below the histogram. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by reverse transcription-coupled quantitative PCR (RT-qPCR), with specific primers for the human β-globin pre-mRNA, as shown in (A). For each case, intron 1 and intron 2 containing human β-globin pre-RNAs levels were determined by normalization to the level of murine glyceraldehyde 3-phosphate dehydrogenase (GAPDH) 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 using Student's t test (unpaired, two-tailed). (C) Schematic representation of the studied human β-globin mRNAs as in Figures 3A and 4A. The pair of arrows represents the coordinates of the amplicon obtained in the qPCR reactions: exon2–3 amplicon. (D) Human β-globin mRNA quantification was performed by RT-qPCR as in (B), but using specific primers for the human β-globin processed mRNA. Levels of each human β-globin mRNA variant were determined by normalization to the level of murine GAPDH mRNA, using the comparative Ct method, and compared to the wild-type control. The histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed as in (B).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366927&req=5

pone-0038505-g005: The presence of the nonsense codon equally decreases the abundance of intron 1 versus intron 2 containing human β-globin pre-mRNAs.(A) Schematic representation of the human β-globin pre-mRNA, as in Figures 3D and 4D. The two pairs of arrows represent the coordinates of both amplicons obtained in the qPCR reactions: intron1-exon2 and exon2-intron2 amplicons. (B) MEL cells were stably transfected with a test human β-globin construct as specified below the histogram. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by reverse transcription-coupled quantitative PCR (RT-qPCR), with specific primers for the human β-globin pre-mRNA, as shown in (A). For each case, intron 1 and intron 2 containing human β-globin pre-RNAs levels were determined by normalization to the level of murine glyceraldehyde 3-phosphate dehydrogenase (GAPDH) 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 using Student's t test (unpaired, two-tailed). (C) Schematic representation of the studied human β-globin mRNAs as in Figures 3A and 4A. The pair of arrows represents the coordinates of the amplicon obtained in the qPCR reactions: exon2–3 amplicon. (D) Human β-globin mRNA quantification was performed by RT-qPCR as in (B), but using specific primers for the human β-globin processed mRNA. Levels of each human β-globin mRNA variant were determined by normalization to the level of murine GAPDH mRNA, using the comparative Ct method, and compared to the wild-type control. The histogram shows the mean and standard deviations from three independent experiments. Statistical analysis was performed as in (B).
Mentions: In order to test to what extent the presence of the nonsense codon affects the relative amount of intron 1 versus intron 2 containing β-globin pre-mRNAs, we analysed the β39 and β62 transcripts stably expressed in differentiated MEL cell pools and results were compared to those of the βWT, β127 and β39missense control transcripts. This analysis was carried out by reverse transcription-coupled quantitative PCR (RT-qPCR) assays to specifically quantify the amount of either intron 1 or intron 2 containing human β-globin pre-mRNAs (Figure 5). Thus, pre-mRNA quantification was carried out with two sets of primers specific for the human β-globin intron 1 and intron 2 pre-mRNA sequence, respectively, using a set of primers specific for the murine GAPDH mRNA as an internal control (Figure 5A, B). As a control, RT-qPCR was also performed with a set of specific primers to quantify processed mRNA, to show that, under these experimental conditions, the PTCs at position 39 or 62 are able to induce a strong downregulation of the steady-state levels as expected for mRNAs typically committed to NMD, while levels of mRNA bearing a PTC at the 3′-most exon (β127) are not significantly different from the normal control (Figure 5C, D). The quantitative PCR efficiency for all amplicons was found to be similar and near to 100%. Control reactions using total RNA samples from untransfected MEL cells, confirmed that unspecific amplification of the murine β-globin transcripts was negligible. In agreement with the previously obtained RPA data, RT-qPCR analysis of the intron 2-containing pre-mRNA steady-state levels shows a significant 2.3 to 3.8-fold reduction of the β39 and β62 unspliced RNAs relatively to the βWT pre-mRNA (P<0.01) (Figure 5B). On the other hand, β127 and β39missense unspliced transcripts exhibit similar levels, which are not significantly different from the normal control (P = 0.12 and P = 0.08, respectively). Additionally, in each case, both β-globin intron 1 and intron 2 containing pre-mRNAs yielded very similar expression levels (P>0.05). Therefore, the presence of the NMD-sensitive nonsense codons does not differentially affect the rates of removal of intron 1 and 2, and, thus, splicing efficiency in transcripts bearing NMD-competent nonsense codons seems to be normal.

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