Nonsense-mediated translational repression involves exon junction complex downstream of premature translation termination codon

Abstract We have analyzed a cloned beta O-thalassemia (beta O-thal) gene from a patient doubly heterozygous for hemoglobin Lepore and beta O- thalassemia. Studies of 3H-uridine incorporation into beta-globin mRNA in this patient's erythroblasts suggested an intranuclear defect in both beta and Lepore (delta beta) mRNA synthesis, as did S1 nuclease analysis of nuclear RNA. However, the nucleotide sequence of the beta O- thal gene revealed only a single base change in codon 39 (CAG----UAG), which created a premature translation termination codon. The 5′ flanking sequence, including transcription promotor boxes and the mRNA initiation (CAP) site, were normal. The unexpected effect of this mutation on intranuclear beta-mRNA synthesis in vivo was studied by insertion of the cloned gene into a plasmid expression vector and transfection into tissue culture (COS-1) cells. beta-Globin mRNA produced by the transfected cells was assessed by S1 nuclease analysis. The beta O-39 thalassemia gene generated five- to tenfold less beta- mRNA than a normal beta-gene in both nuclear and cytoplasmic RNA, simulating the results observed in vivo. Moreover, the small amount of beta O-39 mRNA produced was as stable as normal beta-mRNA during an actinomycin D chase, ruling out rapid cytoplasmic turnover as a cause of the reduced accumulation. Cotransfection of the beta O-39 thalassemia gene with a mutant tyrosine suppressor tRNA gene resulted in restoration of the beta O-39 mRNA accumulation to near-normal levels. On the basis of these results, we suggest that the low levels of beta-mRNA known to exist in the common form of beta O-thalassemia, beta O-39 thalassemia, result from a lesion in transcription, or early posttranscriptional processes; the defect appears to be corrected by restoration of proper translational potential to the mutant mRNA, at least in a gene transfer-expression system in tissue-culture cells.

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Intranuclear defect in beta-globin mRNA accumulation due to a premature translation termination codon

Blood ◽

10.1182/blood.v64.1.13.bloodjournal64113 ◽

1984 ◽

Vol 64 (1) ◽

pp. 13-22

Author(s):

K Takeshita ◽

BG Forget ◽

A Scarpa ◽

EJ Jr Benz

Keyword(s):

Translation Termination ◽

Beta Globin ◽

Mrna Synthesis ◽

Globin Mrna ◽

Mrna Accumulation ◽

S1 Nuclease ◽

Translation Termination Codon ◽

Premature Translation Termination ◽

Premature Translation Termination Codon

We have analyzed a cloned beta O-thalassemia (beta O-thal) gene from a patient doubly heterozygous for hemoglobin Lepore and beta O- thalassemia. Studies of 3H-uridine incorporation into beta-globin mRNA in this patient's erythroblasts suggested an intranuclear defect in both beta and Lepore (delta beta) mRNA synthesis, as did S1 nuclease analysis of nuclear RNA. However, the nucleotide sequence of the beta O- thal gene revealed only a single base change in codon 39 (CAG----UAG), which created a premature translation termination codon. The 5′ flanking sequence, including transcription promotor boxes and the mRNA initiation (CAP) site, were normal. The unexpected effect of this mutation on intranuclear beta-mRNA synthesis in vivo was studied by insertion of the cloned gene into a plasmid expression vector and transfection into tissue culture (COS-1) cells. beta-Globin mRNA produced by the transfected cells was assessed by S1 nuclease analysis. The beta O-39 thalassemia gene generated five- to tenfold less beta- mRNA than a normal beta-gene in both nuclear and cytoplasmic RNA, simulating the results observed in vivo. Moreover, the small amount of beta O-39 mRNA produced was as stable as normal beta-mRNA during an actinomycin D chase, ruling out rapid cytoplasmic turnover as a cause of the reduced accumulation. Cotransfection of the beta O-39 thalassemia gene with a mutant tyrosine suppressor tRNA gene resulted in restoration of the beta O-39 mRNA accumulation to near-normal levels. On the basis of these results, we suggest that the low levels of beta-mRNA known to exist in the common form of beta O-thalassemia, beta O-39 thalassemia, result from a lesion in transcription, or early posttranscriptional processes; the defect appears to be corrected by restoration of proper translational potential to the mutant mRNA, at least in a gene transfer-expression system in tissue-culture cells.

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An intron proximal to a PTC enhances NMD in Saccharomyces cerevisiae

10.1101/149245 ◽

2017 ◽

Author(s):

Jikai Wen ◽

Muyang He ◽

Marija Petric ◽

Laetitia Marzi ◽

Jianming Wang ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Mammalian Cells ◽

Mrna Decay ◽

Translation Termination ◽

Exon Junction Complex ◽

Coding Region ◽

Independent Mechanism ◽

Nonsense Mediated Mrna Decay ◽

Translation Termination Codon ◽

Premature Translation Termination Codon

AbstractNonsense mediated mRNA decay (NMD) is regarded as the function of a specialized cytoplasmic translation-coupled mRNA decay pathway in eukaryotes, however, whether a premature translation termination codon (PTC) will lead to NMD often depends on splicing a downstream intron in the nucleus. Deposition of the exon junction complex (EJC) on mRNA is understood to mediate such splicing-dependent NMD in mammalian cells. The budding yeast, Saccharomyces cerevisiae, which has introns in only 5% of its genes, characteristically at the start of the coding region, and lacks proteins essential for EJC assembly, is not expected to undergo splicing-dependent NMD. However, we found that the presence of an intron near a PTC can also enhance NMD in this organism, regardless of whether it is downstream or upstream. These data provide evidence for a hitherto unsuspected EJC-independent mechanism linking translation and pre-mRNA in S. cerevisiae.

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Resolution of the discrepancy between a gene translation - termination codon and the deduced sequence for release of the encoded polypeptide

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1983.tb07855.x ◽

1983 ◽

Vol 137 (3) ◽

pp. 509-516 ◽

Cited By ~ 4

Author(s):

John F. ATKINS ◽

Raymond F. GESTELAND

Keyword(s):

Translation Termination ◽

Termination Codon ◽

Translation Termination Codon

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A role for DIS3L2 over human nonsense-mediated mRNA decay targets

10.1101/722702 ◽

2019 ◽

Cited By ~ 1

Author(s):

Paulo J. da Costa ◽

Juliane Menezes ◽

Margarida Saramago ◽

Juan F. García-Moreno ◽

Hugo A. Santos ◽

...

Keyword(s):

Translation Termination ◽

Full Length ◽

Nonsense Mediated Decay ◽

Endonucleolytic Cleavage ◽

Nonsense Mediated Mrna Decay ◽

Translation Termination Codon ◽

Life Analysis ◽

Mrna Half Life ◽

Premature Translation Termination Codon

ABSTRACTThe nonsense-mediated decay (NMD) pathway selectively degrades mRNAs carrying a premature translation-termination codon but also regulates the abundance of a large number of physiological mRNAs that encode full-length proteins. In human cells, NMD-targeted mRNAs are degraded by endonucleolytic cleavage and exonucleolytic degradation from both 5’ and 3’ ends. This is done by a process not yet completely understood that recruits decapping and 5’-to-3’ exonuclease activities, as well as deadenylating and 3’-to-5’ exonuclease exosome activities. In yeast, DIS3/Rrp44 protein is the catalytic subunit of the exosome, but in humans, there are three known paralogues of this enzyme: DIS3, DIS3L1, and DIS3L2. DIS3L1 and DIS3L2 exoribonucleases localize in the same compartment where NMD occurs, but little is known about their role in this process. In order to unveil the role of DIS3L2 in NMD, here we show that some NMD-targets accumulate in DIS3L2-depleted cells. mRNA half-life analysis further supports that these NMD-targets are in fact DIS3L2 substrates. Besides, we observed that DIS3L2 acts over full-length transcripts, through a process that also involves UPF1. Moreover, DIS3L2-mediated decay is dependent on the activity of the terminal uridylyl transferases Zcchc6/11 (TUT7/4). Together, our findings establish a role for DIS3L2 and uridylation in NMD.

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Mistranslation of a TGA termination codon as tryptophan in recombinant platelet-derived growth factor expressed in Escherichia coli

Biochemical Journal ◽

10.1042/bj3090411 ◽

1995 ◽

Vol 309 (2) ◽

pp. 411-417 ◽

Cited By ~ 11

Author(s):

K V Lu ◽

M F Rohde ◽

A R Thomason ◽

W C Kenney ◽

H S Lu

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Amino Acid ◽

Growth Factor ◽

Translation Termination ◽

Termination Codon ◽

Platelet Derived Growth Factor ◽

E Coli ◽

Translation Termination Codon ◽

Terminal Amino

The mature 109-amino-acid human platelet-derived growth factor B (PDGF-B) peptide is derived by intracellular processing from a 241-amino-acid precursor synthesized in mammalian cells, with removal of 81 N-terminal and 51 C-terminal amino acids. In order to produce directly the mature 109-amino acid PDGF-B peptide as a recombinant protein in Escherichia coli, a CGA codon at position 110 of a DNA sequence encoding the full-length precursor form of PDGF-B was converted into the translation termination codon TGA by in vitro mutagenesis. Expression of this DNA via a plasmid vector in E. coli resulted in production of two distinct PDGF-B proteins having apparent molecular masses of 15 and 19 kDa, with the latter species predominating. Structural characterization employing N- and C-terminal amino acid sequencing and MS analyses indicated that the 15 kDa protein is the expected 109-amino-acid PDGF-B, and that the 19 kDa protein represents a C-terminal extended PDGF-B containing 160 amino acids. Characterization of a unique tryptic peptide derived from the 19 kDa protein revealed that this longer form of PDGF-B results from mistranslation of the introduced TGA termination codon at position 110 as tryptophan, with translation subsequently proceeding to the naturally occurring TAG termination codon at position 161. Owing to the high rate of translation readthrough of TGA codons in this and occasionally other proteins, it appears that the use of TGA as a translation termination codon for proteins to be expressed in E. coli should be avoided when possible.

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Translation Termination Is Involved in Histone mRNA Degradation when DNA Replication Is Inhibited

Molecular and Cellular Biology ◽

10.1128/mcb.25.16.6879-6888.2005 ◽

2005 ◽

Vol 25 (16) ◽

pp. 6879-6888 ◽

Cited By ~ 43

Author(s):

Handan Kaygun ◽

William F. Marzluff

Keyword(s):

Dna Synthesis ◽

Translation Termination ◽

Termination Codon ◽

Histone Mrna ◽

Stem Loop ◽

Mrna Metabolism ◽

Histone Mrnas ◽

Iron Response Element ◽

Translation Termination Codon ◽

The Stability

ABSTRACT The levels of replication-dependent histone mRNAs are coordinately regulated with DNA synthesis. A major regulatory step in histone mRNA metabolism is regulation of the half-life of histone mRNAs. Replication-dependent histone mRNAs are the only metazoan mRNAs that are not polyadenylated. Instead, they end with a conserved stem-loop structure, which is recognized by the stem-loop binding protein (SLBP). SLBP is required for histone mRNA processing, as well as translation. We show here, using histone mRNAs whose translation can be regulated by the iron response element, that histone mRNAs need to be actively translated for their rapid degradation following the inhibition of DNA synthesis. We also demonstrate the requirement for translation using a mutant SLBP which is inactive in translation. Histone mRNAs are not rapidly degraded when DNA synthesis is inhibited or at the end of S phase in cells expressing this mutant SLBP. Replication-dependent histone mRNAs have very short 3′ untranslated regions, with the stem-loop located 30 to 70 nucleotides downstream of the translation termination codon. We show here that the stability of histone mRNAs can be modified by altering the position of the stem-loop, thereby changing the distance from the translation termination codon.

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