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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 decreased β-globin pre-mRNA levels are specific for transcripts carrying NMD-competent nonsense mutations.(A) Schematic representation of the test human β-globin mRNA stably expressed in MEL cell pools. The closed and open rectangles depict exons and untranslated regions, respectively. The vertical small arrows represent the position of the nonsense mutations at codon 26 (GAG→UAG; β26), 39 (CAG→UAG; β39), 62 (GCT→UAG; β62) or 127 (CAG→UAG; β127). Position of initiation (AUG) and termination (UAA) codons, as well as cap structure (m7G) and poly(A) tail [(A)n] are also represented. Localization and length in nucleotides (nt) of the probe comprising intron 2-exon 3 sequences (βintron2exon3 probe) for detection and quantification of the human β-globin RNA by ribonuclease protection assays (RPA) is shown below the diagram. (B) MEL cells were stably transfected with a test human β-globin construct as specified above each lane. A 2-fold RNA sample (βWT×2) from MEL cells transfected with the βWT gene was also assayed to demonstrate that the experimental RPA was carried out in probe excess. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β- and mouse α-globin mRNAs (see Materials and Methods). The protected bands corresponding to the human β-globin and mouse α-globin mRNAs are shown on the right, and the corresponding intensities were quantified by phosphorimaging. The level of mRNA from each β-globin allele was normalized to the level of endogenous mouse α-globin in order to control for RNA recovery and erythroid differentiation induction. Normalized values were then calculated as the percentage of wild-type β-globin mRNA. (C) The percentage mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (D) Schematic representation of the test human β-globin pre-mRNA stably expressed in MEL cell pools as in (A). Localization and length in nucleotides (nt) of the probe comprising part of intron 2 (βintron2 probe) for detection and quantification of the human β-globin pre-mRNA by RPA is shown below the diagram. (E) After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β-globin pre-mRNA and mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (see Materials and Methods). The corresponding protected bands are shown on the right, and their intensities were quantified by phosphorimaging as in (B). (F) The percentage pre-mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown, as in (C). Statistical analysis was performed using Student's t test (unpaired, two-tailed).
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pone-0038505-g004: The decreased β-globin pre-mRNA levels are specific for transcripts carrying NMD-competent nonsense mutations.(A) Schematic representation of the test human β-globin mRNA stably expressed in MEL cell pools. The closed and open rectangles depict exons and untranslated regions, respectively. The vertical small arrows represent the position of the nonsense mutations at codon 26 (GAG→UAG; β26), 39 (CAG→UAG; β39), 62 (GCT→UAG; β62) or 127 (CAG→UAG; β127). Position of initiation (AUG) and termination (UAA) codons, as well as cap structure (m7G) and poly(A) tail [(A)n] are also represented. Localization and length in nucleotides (nt) of the probe comprising intron 2-exon 3 sequences (βintron2exon3 probe) for detection and quantification of the human β-globin RNA by ribonuclease protection assays (RPA) is shown below the diagram. (B) MEL cells were stably transfected with a test human β-globin construct as specified above each lane. A 2-fold RNA sample (βWT×2) from MEL cells transfected with the βWT gene was also assayed to demonstrate that the experimental RPA was carried out in probe excess. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β- and mouse α-globin mRNAs (see Materials and Methods). The protected bands corresponding to the human β-globin and mouse α-globin mRNAs are shown on the right, and the corresponding intensities were quantified by phosphorimaging. The level of mRNA from each β-globin allele was normalized to the level of endogenous mouse α-globin in order to control for RNA recovery and erythroid differentiation induction. Normalized values were then calculated as the percentage of wild-type β-globin mRNA. (C) The percentage mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (D) Schematic representation of the test human β-globin pre-mRNA stably expressed in MEL cell pools as in (A). Localization and length in nucleotides (nt) of the probe comprising part of intron 2 (βintron2 probe) for detection and quantification of the human β-globin pre-mRNA by RPA is shown below the diagram. (E) After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β-globin pre-mRNA and mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (see Materials and Methods). The corresponding protected bands are shown on the right, and their intensities were quantified by phosphorimaging as in (B). (F) The percentage pre-mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown, as in (C). Statistical analysis was performed using Student's t test (unpaired, two-tailed).

Mentions: Considering the formerly observed downregulation of unspliced β-globin transcripts carrying a nonsense mutation at codon 39, we next asked whether this effect occurs in other transcripts carrying a different PTC. We thus established two different MEL cell pools stably expressing the human β-globin gene carrying a NMD-sensitive nonsense mutation at codon 26 (exon 1; β26) or at codon 62 (exon 2; β62) [36]. The β26 and β62 mRNAs were previously found to accumulate at reduced steady-state levels when compared to the wild-type β-globin mRNA in erythroid and non-erythroid cells [32], [36] These transcripts are NMD-sensitive because the respective PTCs are located more than 50–54 nts upstream to the 3′-most exon-exon junction and when the ribosome reaches the PTC, the terminating complex can interact with the downstream EJC via UPF1 [36], [37]. Regarding β62 MEL cell pools, we were able to isolate two independent MEL cell pools (β62#1 and β62#2). After erythroid cell differentiation induction, the transgene mRNA levels were determined by RPA as before, using probes comprising part of the human β-globin intron 2 and exon 3 or murine α-globin mRNA sequences, and results were compared to those of MEL cell pools stably expressing the βWT or β39 genes (Figure 4A, B). According to our previously published data [36], our results show that β26 and β62 mRNA levels of the corresponding MEL cell pools are strongly downregulated relatively to the βWT mRNA levels, presenting levels similar to those observed in the β39 MEL cell pools, meaning that they are induced to rapid decay, as expected (Figure 4B, C). These results indicate that under our experimental conditions, the cellular NMD machinery is functional.


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 decreased β-globin pre-mRNA levels are specific for transcripts carrying NMD-competent nonsense mutations.(A) Schematic representation of the test human β-globin mRNA stably expressed in MEL cell pools. The closed and open rectangles depict exons and untranslated regions, respectively. The vertical small arrows represent the position of the nonsense mutations at codon 26 (GAG→UAG; β26), 39 (CAG→UAG; β39), 62 (GCT→UAG; β62) or 127 (CAG→UAG; β127). Position of initiation (AUG) and termination (UAA) codons, as well as cap structure (m7G) and poly(A) tail [(A)n] are also represented. Localization and length in nucleotides (nt) of the probe comprising intron 2-exon 3 sequences (βintron2exon3 probe) for detection and quantification of the human β-globin RNA by ribonuclease protection assays (RPA) is shown below the diagram. (B) MEL cells were stably transfected with a test human β-globin construct as specified above each lane. A 2-fold RNA sample (βWT×2) from MEL cells transfected with the βWT gene was also assayed to demonstrate that the experimental RPA was carried out in probe excess. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β- and mouse α-globin mRNAs (see Materials and Methods). The protected bands corresponding to the human β-globin and mouse α-globin mRNAs are shown on the right, and the corresponding intensities were quantified by phosphorimaging. The level of mRNA from each β-globin allele was normalized to the level of endogenous mouse α-globin in order to control for RNA recovery and erythroid differentiation induction. Normalized values were then calculated as the percentage of wild-type β-globin mRNA. (C) The percentage mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (D) Schematic representation of the test human β-globin pre-mRNA stably expressed in MEL cell pools as in (A). Localization and length in nucleotides (nt) of the probe comprising part of intron 2 (βintron2 probe) for detection and quantification of the human β-globin pre-mRNA by RPA is shown below the diagram. (E) After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β-globin pre-mRNA and mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (see Materials and Methods). The corresponding protected bands are shown on the right, and their intensities were quantified by phosphorimaging as in (B). (F) The percentage pre-mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown, as in (C). Statistical analysis was performed using Student's t test (unpaired, two-tailed).
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Related In: Results  -  Collection

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

pone-0038505-g004: The decreased β-globin pre-mRNA levels are specific for transcripts carrying NMD-competent nonsense mutations.(A) Schematic representation of the test human β-globin mRNA stably expressed in MEL cell pools. The closed and open rectangles depict exons and untranslated regions, respectively. The vertical small arrows represent the position of the nonsense mutations at codon 26 (GAG→UAG; β26), 39 (CAG→UAG; β39), 62 (GCT→UAG; β62) or 127 (CAG→UAG; β127). Position of initiation (AUG) and termination (UAA) codons, as well as cap structure (m7G) and poly(A) tail [(A)n] are also represented. Localization and length in nucleotides (nt) of the probe comprising intron 2-exon 3 sequences (βintron2exon3 probe) for detection and quantification of the human β-globin RNA by ribonuclease protection assays (RPA) is shown below the diagram. (B) MEL cells were stably transfected with a test human β-globin construct as specified above each lane. A 2-fold RNA sample (βWT×2) from MEL cells transfected with the βWT gene was also assayed to demonstrate that the experimental RPA was carried out in probe excess. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β- and mouse α-globin mRNAs (see Materials and Methods). The protected bands corresponding to the human β-globin and mouse α-globin mRNAs are shown on the right, and the corresponding intensities were quantified by phosphorimaging. The level of mRNA from each β-globin allele was normalized to the level of endogenous mouse α-globin in order to control for RNA recovery and erythroid differentiation induction. Normalized values were then calculated as the percentage of wild-type β-globin mRNA. (C) The percentage mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (D) Schematic representation of the test human β-globin pre-mRNA stably expressed in MEL cell pools as in (A). Localization and length in nucleotides (nt) of the probe comprising part of intron 2 (βintron2 probe) for detection and quantification of the human β-globin pre-mRNA by RPA is shown below the diagram. (E) After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β-globin pre-mRNA and mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (see Materials and Methods). The corresponding protected bands are shown on the right, and their intensities were quantified by phosphorimaging as in (B). (F) The percentage pre-mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown, as in (C). Statistical analysis was performed using Student's t test (unpaired, two-tailed).
Mentions: Considering the formerly observed downregulation of unspliced β-globin transcripts carrying a nonsense mutation at codon 39, we next asked whether this effect occurs in other transcripts carrying a different PTC. We thus established two different MEL cell pools stably expressing the human β-globin gene carrying a NMD-sensitive nonsense mutation at codon 26 (exon 1; β26) or at codon 62 (exon 2; β62) [36]. The β26 and β62 mRNAs were previously found to accumulate at reduced steady-state levels when compared to the wild-type β-globin mRNA in erythroid and non-erythroid cells [32], [36] These transcripts are NMD-sensitive because the respective PTCs are located more than 50–54 nts upstream to the 3′-most exon-exon junction and when the ribosome reaches the PTC, the terminating complex can interact with the downstream EJC via UPF1 [36], [37]. Regarding β62 MEL cell pools, we were able to isolate two independent MEL cell pools (β62#1 and β62#2). After erythroid cell differentiation induction, the transgene mRNA levels were determined by RPA as before, using probes comprising part of the human β-globin intron 2 and exon 3 or murine α-globin mRNA sequences, and results were compared to those of MEL cell pools stably expressing the βWT or β39 genes (Figure 4A, B). According to our previously published data [36], our results show that β26 and β62 mRNA levels of the corresponding MEL cell pools are strongly downregulated relatively to the βWT mRNA levels, presenting levels similar to those observed in the β39 MEL cell pools, meaning that they are induced to rapid decay, as expected (Figure 4B, C). These results indicate that under our experimental conditions, the cellular NMD machinery is functional.

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