Unspliced Precursors of NMD-Sensitive β-Globin Transcripts Exhibit Decreased Steady-State Levels in Erythroid Cells.

Ana Morgado, Fátima Almeida, Alexandre Teixeira, Ana Luísa Silva, Luísa Romão

Research output: Contribution to journalArticle

3 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)Online
JournalPLoS ONE
Volume7
Issue number6
DOIs
Publication statusPublished - 1 Jan 2012

Fingerprint

Nonsense Mediated mRNA Decay
Erythroid Cells
Globins
Nonsense Codon
stop codon
translation (genetics)
deterioration
RNA Precursors
Messenger RNA
cells
Leukemia, Erythroblastic, Acute
codons
HeLa Cells
reverse transcription
ribonucleases
mice
ribosomes
RNA Stability
Transcription
Ribonucleases

Cite this

Morgado, Ana ; Almeida, Fátima ; Teixeira, Alexandre ; Silva, Ana Luísa ; Romão, Luísa. / Unspliced Precursors of NMD-Sensitive β-Globin Transcripts Exhibit Decreased Steady-State Levels in Erythroid Cells. In: PLoS ONE. 2012 ; Vol. 7, No. 6. pp. Online.
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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.",
keywords = "NONSENSE-MEDIATED DECAY, EXON JUNCTION COMPLEX, GENE-EXPRESSION, POLYMERASE-II, POLY(A)-BINDING PROTEIN, MAMMALIAN-CELLS, MESSENGER-RNA DECAY, PREMATURE TERMINATION CODON, OPEN READING FRAME, QUALITY-CONTROL",
author = "Ana Morgado and F{\'a}tima Almeida and Alexandre Teixeira and Silva, {Ana Lu{\'i}sa} and Lu{\'i}sa Rom{\~a}o",
note = "info:eu-repo/grantAgreement/FCT/SFRH/SFRH{\%}2FBD{\%}2F31920{\%}2F2006/PT# info:eu-repo/grantAgreement/FCT/SFRH/SFRH{\%}2FBD{\%}2F8351{\%}2F2002/PT# This work was partially supported by Fundac¸a˜o para a Cieˆncia e a Tecnologia [Programa de Financiamento Plurianual do Centro de Investigac¸a˜o em Gene´tica Molecular Humana (CIGMH) and Center for Biodiversity, Functional and Integrative Genomics (BioFIG)]. AM, FA and ALS were supported by fellowships from Fundac¸a˜o para a Cieˆncia e a Tecnologia (SFRH/BD/31920/2006, PRAXIS XXI/BPD/18880/98 and SFRH/BD/8351/2002, respectively). No additional external funding was received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip.",
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Unspliced Precursors of NMD-Sensitive β-Globin Transcripts Exhibit Decreased Steady-State Levels in Erythroid Cells. / Morgado, Ana; Almeida, Fátima ; Teixeira, Alexandre ; Silva, Ana Luísa; Romão, Luísa.

In: PLoS ONE, Vol. 7, No. 6, 01.01.2012, p. Online.

Research output: Contribution to journalArticle

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T1 - Unspliced Precursors of NMD-Sensitive β-Globin Transcripts Exhibit Decreased Steady-State Levels in Erythroid Cells.

AU - Morgado, Ana

AU - Almeida, Fátima

AU - Teixeira, Alexandre

AU - Silva, Ana Luísa

AU - Romão, Luísa

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Y1 - 2012/1/1

N2 - 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.

AB - 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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KW - EXON JUNCTION COMPLEX

KW - GENE-EXPRESSION

KW - POLYMERASE-II

KW - POLY(A)-BINDING PROTEIN

KW - MAMMALIAN-CELLS

KW - MESSENGER-RNA DECAY

KW - PREMATURE TERMINATION CODON

KW - OPEN READING FRAME

KW - QUALITY-CONTROL

U2 - 10.1371/journal.pone.0038505

DO - 10.1371/journal.pone.0038505

M3 - Article

VL - 7

SP - Online

JO - PlosOne

JF - PlosOne

SN - 1932-6203

IS - 6

ER -