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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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Human β-globin transgene integrity and copy number analysis in stably transfected MEL cell clones.(A) Representative Southern blot analysis of DNA from MEL cell clones stably transfected with wild-type (βWT) or nonsense-mutated (β39; CAG→UAG) human β-globin gene constructs. Genomic DNA was extracted from MEL cells transfected with a β-globin construct as specified above each lane, where each number indicates an independent cell clone. Untransfected MEL (t- MEL) and human genomic DNA were used as negative and positive controls, respectively. DNA was digested with EcoNI plus KpnI enzymes and blots were hybridized with a [α-32P]dCTP-labeled probe of the human β-globin gene that recognizes a 5.0 kb fragment integrated in the murine genome or a 6.1 kb fragment in the human genomic DNA. MEL cell clones selected for further analysis are indicated in bold. (B) Transgene copy number for each selected MEL cell clone was determined by quantitative PCR using primers specific for human β-globin gene and the endogenous murine Thy1 gene. Quantification was performed by the relative standard curve method. Chart shows the mean ± standard deviation qPCR data from three independent experiments.
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pone-0038505-g001: Human β-globin transgene integrity and copy number analysis in stably transfected MEL cell clones.(A) Representative Southern blot analysis of DNA from MEL cell clones stably transfected with wild-type (βWT) or nonsense-mutated (β39; CAG→UAG) human β-globin gene constructs. Genomic DNA was extracted from MEL cells transfected with a β-globin construct as specified above each lane, where each number indicates an independent cell clone. Untransfected MEL (t- MEL) and human genomic DNA were used as negative and positive controls, respectively. DNA was digested with EcoNI plus KpnI enzymes and blots were hybridized with a [α-32P]dCTP-labeled probe of the human β-globin gene that recognizes a 5.0 kb fragment integrated in the murine genome or a 6.1 kb fragment in the human genomic DNA. MEL cell clones selected for further analysis are indicated in bold. (B) Transgene copy number for each selected MEL cell clone was determined by quantitative PCR using primers specific for human β-globin gene and the endogenous murine Thy1 gene. Quantification was performed by the relative standard curve method. Chart shows the mean ± standard deviation qPCR data from three independent experiments.

Mentions: With the aim to investigate if the presence of a nonsense codon in a transcript could affect its nuclear metabolism, in this study, we generated stably transfected MEL cell clones expressing the wild-type human β-globin gene (βWT), or a β-globin gene variant carrying a nonsense mutation at codon 39 (β39), which is a well-characterized β-globin NMD substrate in erythroid as well as in non-erythroid cells [32]–[35]. Each human β-globin gene was cloned into the p158.2 vector, as previously described, where it is expressed under the transcriptional control of the corresponding promoter and the DNase I hypersensitive site 2 of the human locus control region [32]. To select cell line clones for further studies, the integration of the intact human β-globin gene in the murine genome was evaluated by Southern blot analysis (Figure 1A). From the different clones analysed, we have chosen, for further analyses, six independent clones – #146, #154, #166 expressing the βWT gene, and #241, #249 and #252 expressing the β39 gene. The chosen cell clones show the same pattern of integration and different copy number of integrated transgenes. In these selected clones, accurate evaluation of the human β-globin transgene copy number was performed by quantitative PCR using the endogenous diploid thymus cell antigen (Thy1) gene as a copy number reference. Results have shown that for the three βWT independent selected clones, the corresponding transgene copy number is 18±2, 24±3 and 34±5, whereas in the β39 cell lines, the transgene copy number is 22±6, 30±1 and 18±2 (Figure 1B). This selection allowed us to perform further gene expression analyses in pairs of βWT and β39 clones matched for transgene copy number.


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)

Human β-globin transgene integrity and copy number analysis in stably transfected MEL cell clones.(A) Representative Southern blot analysis of DNA from MEL cell clones stably transfected with wild-type (βWT) or nonsense-mutated (β39; CAG→UAG) human β-globin gene constructs. Genomic DNA was extracted from MEL cells transfected with a β-globin construct as specified above each lane, where each number indicates an independent cell clone. Untransfected MEL (t- MEL) and human genomic DNA were used as negative and positive controls, respectively. DNA was digested with EcoNI plus KpnI enzymes and blots were hybridized with a [α-32P]dCTP-labeled probe of the human β-globin gene that recognizes a 5.0 kb fragment integrated in the murine genome or a 6.1 kb fragment in the human genomic DNA. MEL cell clones selected for further analysis are indicated in bold. (B) Transgene copy number for each selected MEL cell clone was determined by quantitative PCR using primers specific for human β-globin gene and the endogenous murine Thy1 gene. Quantification was performed by the relative standard curve method. Chart shows the mean ± standard deviation qPCR data from three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366927&req=5

pone-0038505-g001: Human β-globin transgene integrity and copy number analysis in stably transfected MEL cell clones.(A) Representative Southern blot analysis of DNA from MEL cell clones stably transfected with wild-type (βWT) or nonsense-mutated (β39; CAG→UAG) human β-globin gene constructs. Genomic DNA was extracted from MEL cells transfected with a β-globin construct as specified above each lane, where each number indicates an independent cell clone. Untransfected MEL (t- MEL) and human genomic DNA were used as negative and positive controls, respectively. DNA was digested with EcoNI plus KpnI enzymes and blots were hybridized with a [α-32P]dCTP-labeled probe of the human β-globin gene that recognizes a 5.0 kb fragment integrated in the murine genome or a 6.1 kb fragment in the human genomic DNA. MEL cell clones selected for further analysis are indicated in bold. (B) Transgene copy number for each selected MEL cell clone was determined by quantitative PCR using primers specific for human β-globin gene and the endogenous murine Thy1 gene. Quantification was performed by the relative standard curve method. Chart shows the mean ± standard deviation qPCR data from three independent experiments.
Mentions: With the aim to investigate if the presence of a nonsense codon in a transcript could affect its nuclear metabolism, in this study, we generated stably transfected MEL cell clones expressing the wild-type human β-globin gene (βWT), or a β-globin gene variant carrying a nonsense mutation at codon 39 (β39), which is a well-characterized β-globin NMD substrate in erythroid as well as in non-erythroid cells [32]–[35]. Each human β-globin gene was cloned into the p158.2 vector, as previously described, where it is expressed under the transcriptional control of the corresponding promoter and the DNase I hypersensitive site 2 of the human locus control region [32]. To select cell line clones for further studies, the integration of the intact human β-globin gene in the murine genome was evaluated by Southern blot analysis (Figure 1A). From the different clones analysed, we have chosen, for further analyses, six independent clones – #146, #154, #166 expressing the βWT gene, and #241, #249 and #252 expressing the β39 gene. The chosen cell clones show the same pattern of integration and different copy number of integrated transgenes. In these selected clones, accurate evaluation of the human β-globin transgene copy number was performed by quantitative PCR using the endogenous diploid thymus cell antigen (Thy1) gene as a copy number reference. Results have shown that for the three βWT independent selected clones, the corresponding transgene copy number is 18±2, 24±3 and 34±5, whereas in the β39 cell lines, the transgene copy number is 22±6, 30±1 and 18±2 (Figure 1B). This selection allowed us to perform further gene expression analyses in pairs of βWT and β39 clones matched for transgene copy number.

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