scholarly journals Infrequent Translation of a Nonsense Codon Is Sufficient to Decrease mRNA Level

1999 ◽  
Vol 10 (3) ◽  
pp. 515-524 ◽  
Author(s):  
Alla Buzina ◽  
Marc J. Shulman
Keyword(s):  
Mammalian Cells ◽  
Translation Termination ◽  
Mrna Level ◽  
Chain Gene ◽  
Nonsense Mutations ◽  
Nonsense Codon ◽  
Residual Level ◽  
Nonsense Codons ◽  
High Level ◽  
In Cis

In many organisms nonsense mutations decrease the level of mRNA. In the case of mammalian cells, it is still controversial whether translation is required for this nonsense-mediated RNA decrease (NMD). Although previous analyzes have shown that conditions that impede translation termination at nonsense codons also prevent NMD, the residual level of termination was unknown in these experiments. Moreover, the conditions used to impede termination might also have interfered with NMD in other ways. Because of these uncertainties, we have tested the effects of limiting translation of a nonsense codon in a different way, using two mutations in the immunoglobulin μ heavy chain gene. For this purpose we exploited an exceptional nonsense mutation at codon 3, which efficiently terminates translation but nonetheless maintains a high level of μ mRNA. We have shown 1) that translation of Ter462 in the double mutant occurs at only ∼4% the normal frequency, and 2) that Ter462 in cis with Ter3 can induce NMD. That is, translation of Ter462 at this low (4%) frequency is sufficient to induce NMD.

scholarly journals At Least One Intron Is Required for the Nonsense-Mediated Decay of Triosephosphate Isomerase mRNA: a Possible Link between Nuclear Splicing and Cytoplasmic Translation

1998 ◽  
Vol 18 (9) ◽  
pp. 5272-5283 ◽  
Author(s):  
Jing Zhang ◽  
Xiaolei Sun ◽  
Yimei Qian ◽  
Jeffrey P. LaDuca ◽  
Lynne E. Maquat
Keyword(s):  
Translation Initiation ◽  
Mammalian Cells ◽  
Mrna Decay ◽  
Triosephosphate Isomerase ◽  
Translation Termination ◽  
Nonsense Mediated Decay ◽  
Nonsense Codon ◽  
Translation Initiation Codon ◽  
Exon Junctions ◽  
Nonsense Codons

ABSTRACT Mammalian cells have established mechanisms to reduce the abundance of mRNAs that harbor a nonsense codon and prematurely terminate translation. In the case of the human triosephosphate isomerase (TPI gene), nonsense codons located less than 50 to 55 bp upstream of intron 6, the 3′-most intron, fail to mediate mRNA decay. With the aim of understanding the feature(s) of TPI intron 6 that confer function in positioning the boundary between nonsense codons that do and do not mediate decay, the effects of deleting or duplicating introns have been assessed. The results demonstrate that TPI intron 6 functions to position the boundary because it is the 3′-most intron. Since decay takes place after pre-mRNA splicing, it is conceivable that removal of the 3′-most intron from pre-mRNA “marks” the 3′-most exon-exon junction of product mRNA so that only nonsense codons located more than 50 to 55 nucleotides upstream of the “mark” mediate mRNA decay. Decay may be elicited by the failure of translating ribosomes to translate sufficiently close to the mark or, more likely, the scanning or looping out of some component(s) of the translation termination complex to the mark. In support of scanning, a nonsense codon does not elicit decay if some of the introns that normally reside downstream of the nonsense codon are deleted so the nonsense codon is located (i) too far away from a downstream intron, suggesting that all exon-exon junctions may be marked, and (ii) too far away from a downstream failsafe sequence that appears to function on behalf of intron 6, i.e., when intron 6 fails to leave a mark. Notably, the proposed scanning complex may have a greater unwinding capability than the complex that scans for a translation initiation codon since a hairpin structure strong enough to block translation initiation when inserted into the 5′ untranslated region does not block nonsense-mediated decay when inserted into exon 6 between a nonsense codon residing in exon 6 and intron 6.

Mammalian nonsense codons can be cis effectors of nuclear mRNA half-life

1994 ◽  
Vol 14 (12) ◽  
pp. 8219-8228
Author(s):  
P Belgrader ◽  
J Cheng ◽  
X Zhou ◽  
L S Stephenson ◽  
L E Maquat
Keyword(s):  
Triosephosphate Isomerase ◽  
Blot Hybridization ◽  
Mrna Level ◽  
Coding Region ◽  
Protein Coding ◽  
Codon Recognition ◽  
Reverse Transcription Pcr ◽  
Nonsense Codon ◽  
Bona Fide ◽  
Nonsense Codons

Frameshift and nonsense mutations within the gene for human triosephosphate isomerase (TPI) that generate a nonsense codon within the first three-fourths of the protein coding region have been found to reduce the abundance of the product mRNA that copurifies with nuclei. The cellular process and location of the nonsense codon-mediated reduction have proven difficult to elucidate for technical reasons. We show here, using electron microscopy to judge the purity of isolated nuclei, that the previously established reduction to 25% of the normal mRNA level is evident for nuclei that are free of detectable cytoplasmic contamination. Therefore, the reduction is likely to be characteristic of bona fide nuclear RNA. Fully spliced nuclear mRNA is identified by Northern (RNA) blot hybridization and a reverse transcription-PCR assay as the species that undergoes decay in experiments that used the human c-fos promoter to elicit a burst and subsequent shutoff of TPI gene transcription upon the addition of serum to serum-deprived cells. Finally, the finding that deletion of a 5' splice site of the TPI gene results predominantly but not exclusively in the removal by splicing (i.e., skipping) of the upstream exon as a part of the flanking introns has been used to demonstrate that decay is specific to those mRNA products that maintain the nonsense codon. This result, together with our previous results that implicate translation by ribosomes and charged tRNAs in the decay mechanism, indicate that nonsense codon recognition takes place after splicing and triggers decay solely in cis. The possibility that decay takes place during the process of mRNA export from the nucleus to the cytoplasm is discussed.

Nonsense mutations in the human β-globin gene lead to unexpected levels of cytoplasmic mRNA accumulation

Blood ◽  
2000 ◽  
Vol 96 (8) ◽  
pp. 2895-2901 ◽  
Author(s):  
Luı́sa Romão ◽  
Ângela Inácio ◽  
Susana Santos ◽  
Madalena Ávila ◽  
Paula Faustino ◽  
...  
Keyword(s):  
Globin Gene ◽  
Mrna Level ◽  
Erythroid Differentiation ◽  
Mrna Levels ◽  
Globin Genes ◽  
Mrna Accumulation ◽  
Nonsense Mutations ◽  
Differentiation Induction ◽  
Nonsense Codons ◽  
Murine Erythroleukemia

Generally, nonsense codons 50 bp or more upstream of the 3′-most intron of the human β-globin gene reduce mRNA abundance. In contrast, dominantly inherited β-thalassemia is frequently associated with nonsense mutations in the last exon. In this work, murine erythroleukemia (MEL) cells were stably transfected with human β-globin genes mutated within each of the 3 exons, namely at codons 15 (TGG→TGA), 39 (C→T), or 127 (C→T). Primer extension analysis after erythroid differentiation induction showed codon 127 (C→T) mRNA accumulated in the cytoplasm at approximately 20% of the normal mRNA level. Codon 39 (C→T) mutation did not result in significant mRNA accumulation. Unexpectedly, codon 15 (TGG→TGA) mRNA accumulated at approximately 90%. Concordant results were obtained when reticulocyte mRNA from 2 carriers for this mutation was studied. High mRNA accumulation of codon 15 nonsense-mutated gene was revealed to be independent of the type of nonsense mutation and the genomic background in which this mutation occurs. To investigate the effects of other nonsense mutations located in the first exon on the mRNA level, nonsense mutations at codons 5, 17, and 26 were also cloned and stably transfected into MEL cells. After erythroid differentiation induction, mRNAs with a mutation at codon 5 or 17 were detected at high levels, whereas the mutation at codon 26 led to low mRNA levels. These findings suggest that nonsense-mediated mRNA decay is not exclusively dependent on the localization of mutations relative to the 3′-most intron. Other factors may also contribute to determine the cytoplasmic nonsense-mutated mRNA level in erythroid cells.

scholarly journals Introns are cis effectors of the nonsense-codon-mediated reduction in nuclear mRNA abundance.

1994 ◽  
Vol 14 (9) ◽  
pp. 6317-6325 ◽  
Author(s):  
J Cheng ◽  
P Belgrader ◽  
X Zhou ◽  
L E Maquat
Keyword(s):  
Translation Initiation ◽  
Triosephosphate Isomerase ◽  
Splice Sites ◽  
Nonsense Mutations ◽  
Nonsense Codon ◽  
Exon 1 ◽  
Nonsense Codons ◽  
Intron 2 ◽  
Mediated Reduction ◽  
Downstream Movement

The translation of human triosephosphate isomerase (TPI) mRNA normally terminates at codon 249 within exon 7, the final exon. Frameshift and nonsense mutations of the type that cause translation to terminate prematurely at or upstream of codon 189 within exon 6 reduce the level of nuclear TPI mRNA to 20 to 30% of normal by a mechanism that is not a function of the distance of the nonsense codon from either the translation initiation or termination codon. In contrast, frameshift and nonsense mutations of another type that cause translation to terminate prematurely at or downstream of codon 208, also within exon 6, have no effect on the level of nuclear TPI mRNA. In this work, quantitations of RNA that derived from TPI alleles in which nonsense codons had been generated between codons 189 and 208 revealed that the boundary between the two types of nonsense codons resides between codons 192 and 195. The analysis of TPI gene insertions and deletions indicated that the positional feature differentiating the two types of nonsense codons is the distance of the nonsense codon upstream of intron 6. For example, the movement of intron 6 to a position downstream of its normal location resulted in a concomitant downstream movement of the boundary between the two types of nonsense codons. The analysis of intron 6 mutations indicated that the intron 6 effect is stipulated by the 88 nucleotides residing between the 5' and 3' splice sites. Since the deletion of intron 6 resulted in only partial abrogation of the nonsense codon-mediated reduction in the level of TPI mRNA, other sequences within TPI pre-mRNA must function in the effect. One of these sequences may be intron 2, since the deletion of intron 2 also resulted in partial abrogation of the effect. In experiments that switched introns 2 and 6, the replacement of intron 6 with intron 2 was of no consequence to the effect of a nonsense codon within either exon 1 or exon 6. In contrast, the replacement of intron 2 with intron 6 was inconsequential to the effect of a nonsense codon in exon 6 but resulted in partial abrogation of a nonsense codon in exon 1.

Introduction of UAG, UAA, and UGA nonsense mutations at a specific site in the Escherichia coli chloramphenicol acetyltransferase gene: use in measurement of amber, ochre, and opal suppression in mammalian cells

1986 ◽  
Vol 6 (9) ◽  
pp. 3059-3067
Author(s):  
J P Capone ◽  
J M Sedivy ◽  
P A Sharp ◽  
U L RajBhandary
Keyword(s):  
Escherichia Coli ◽  
Enzymatic Activity ◽  
Mammalian Cells ◽  
Chloramphenicol Acetyltransferase ◽  
Nonsense Suppression ◽  
Trna Genes ◽  
Suppressor Activity ◽  
Nonsense Mutations ◽  
Suppressor Trna ◽  
Nonsense Codons

We have used oligonucleotide-directed site-specific mutagenesis to convert serine codon 27 of the Escherichia coli chloramphenicol acetyltransferase (cat) gene to UAG, UAA, and UGA nonsense codons. The mutant cat genes, under transcriptional control of the Rous sarcoma virus long terminal repeat, were then introduced into mammalian cells by DNA transfection along with UAG, UAA, and UGA suppressor tRNA genes derived from a human serine tRNA. Assay for CAT enzymatic activity in extracts from such cells allowed us to detect and quantitate nonsense suppression in monkey CV-1 cells and mouse NIH3T3 cells. Using such an assay, we provide the first direct evidence that an opal suppressor tRNA gene is functional in mammalian cells. The pattern of suppression of the three cat nonsense mutations in bacteria suggests that the serine at position 27 of CAT can be replaced by a wide variety of amino acids without loss of enzymatic activity. Thus, these mutant cat genes should be generally useful for the quantitation of suppressor activity of suppressor tRNA genes introduced into cells and possibly for the detection of naturally occurring nonsense suppressors.

scholarly journals Mutational Evidence for a Functional Connection between Two Domains of 23S rRNA in Translation Termination

2002 ◽  
Vol 184 (18) ◽  
pp. 5052-5057 ◽  
Author(s):  
Alexey L. Arkov ◽  
Klas O. F. Hedenstierna ◽  
Emanuel J. Murgola
Keyword(s):  
Translation Termination ◽  
23S Rrna ◽  
Functional Connection ◽  
Nonsense Mutations ◽  
Isolation And Characterization ◽  
Nonsense Codons ◽  
Peptidyltransferase Center ◽  
Domain V ◽  
First Time

ABSTRACT Nucleotide 1093 in domain II of Escherichia coli 23S rRNA is part of a highly conserved structure historically referred to as the GTPase center. The mutation G1093A was previously shown to cause readthrough of nonsense codons and high temperature-conditional lethality. Defects in translation termination caused by this mutation have also been demonstrated in vitro. To identify sites in 23S rRNA that may be functionally associated with the G1093 region during termination, we selected for secondary mutations in 23S rRNA that would compensate for the temperature-conditional lethality caused by G1093A. Here we report the isolation and characterization of such a secondary mutation. The mutation is a deletion of two consecutive nucleotides from helix 73 in domain V, close to the peptidyltransferase center. The deletion results in a shortening of the CGCG sequence between positions 2045 and 2048 by two nucleotides to CG. In addition to restoring viability in the presence of G1093A, this deletion dramatically decreased readthrough of UGA nonsense mutations caused by G1093A. An analysis of the amount of mutant rRNA in polysomes revealed that this decrease cannot be explained by an inability of G1093A-containing rRNA to be incorporated into polysomes. Furthermore, the deletion was found to cause UGA readthrough on its own, thereby implicating helix 73 in termination for the first time. These results also indicate the existence of a functional connection between the G1093 region and helix 73 during translation termination.

scholarly journals CFTR mRNAs with nonsense codons are degraded by the SMG6-mediated endonucleolytic pathway

2021 ◽  
Author(s):  
Edward Sanderlin ◽  
Melissa Keenan ◽  
Martin Mense ◽  
Alexey Revenko ◽  
Brett Monia ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Exon Junction Complex ◽  
Transmembrane Conductance Regulator ◽  
Loss Of Function ◽  
Transmembrane Conductance ◽  
Nonsense Mutations ◽  
Nonsense Mediated Decay ◽  
Nonsense Codon ◽  
Translational Readthrough ◽  
Nonsense Codons

Abstract Cystic fibrosis is caused by loss of function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene resulting in severe lung disease. Nearly 10% of cystic fibrosis patients have at least one CFTR allele with a nonsense mutation that generates a nonsense codon in the mRNA. Nonsense mutations can result in significant reduction of gene expression partially due to rapid mRNA degradation through the nonsense-mediated decay (NMD) pathway. It has not been thoroughly investigated which branch of the NMD pathway governs the decay of CFTR mRNAs containing nonsense codons. Here we utilized antisense oligonucleotides targeting NMD factors to evaluate the regulation of nonsense codon-containing CFTR mRNAs by the NMD pathway. Interestingly, we found that CFTR mRNAs with G542X, R1162X, and W1282X nonsense codons require UPF2, UPF3, and exon junction complex proteins for NMD, whereas CFTR mRNAs with the Y122X nonsense codon do not. Furthermore, we demonstrated that all evaluated CFTR mRNAs harboring nonsense codons were degraded by the SMG6-mediated endonucleolytic pathway rather than the SMG5/SMG7-mediated exonucleolytic pathway. Finally, we found that stabilization of CFTR mRNAs by NMD inhibition alone improved functional W1282X protein production, and improved the efficiency of aminoglycoside translational readthrough of CFTR-Y122X, -G542X, and -R1162X mRNAs.

Nonsense codons can reduce the abundance of nuclear mRNA without affecting the abundance of pre-mRNA or the half-life of cytoplasmic mRNA

1993 ◽  
Vol 13 (3) ◽  
pp. 1892-1902 ◽  
Author(s):  
J Cheng ◽  
L E Maquat
Keyword(s):  
Rna Splicing ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Triosephosphate Isomerase ◽  
Detectable Effect ◽  
Nonsense Mutations ◽  
Reading Frame ◽  
Nonsense Codon ◽  
Nonsense Codons ◽  
Mediated Reduction

The abundance of the mRNA for human triosephosphate isomerase (TPI) is decreased to approximately 20% of normal by frameshift and nonsense mutations that cause translation to terminate at a nonsense codon within the first three-fourths of the reading frame. Results of previous studies inhibiting RNA synthesis with actinomycin D suggested that the decrease is not attributable to an increased rate of cytoplasmic mRNA decay. However, the step in TPI RNA metabolism that is altered was not defined, and the use of actinomycin D, in affecting all polymerase II-transcribed genes, could result in artifactual conclusions. In data presented here, the nonsense codon-mediated reduction in the level of TPI mRNA is shown to be characteristic of both nuclear and cytoplasmic fractions of the cell, indicating that the altered metabolic step is nucleus associated. Neither aberrancies in gene transcription nor aberrancies in RNA splicing appear to contribute to the reduction since there were no accompanying changes in the amount of nuclear run-on transcription, the level of any of the six introns in TPI pre-mRNA, or the size of processed mRNA in the nucleus. Deletion of all splice sites that reside downstream of a nonsense codon does not abrogate the reduction, indicating that the reduction takes place independently of the splicing of a downstream intron. Experiments that placed TPI gene expression under the control of the human c-fos promoter, which can be transiently activated by the addition of serum to serum-deprived cells, verified that there is no detectable effect of a nonsense codon on the turnover of cytoplasmic mRNA.

scholarly journals Context effects on misreading and suppression at UAG codons in human cells.

1995 ◽  
Vol 15 (12) ◽  
pp. 6593-6600 ◽  
Author(s):  
M K Phillips-Jones ◽  
L S Hill ◽  
J Atkinson ◽  
R Martin
Keyword(s):  
Mammalian Cells ◽  
Context Effects ◽  
Rank Order ◽  
Site Directed Mutagenesis ◽  
Nonsense Suppression ◽  
Lacz Gene ◽  
Culture Cells ◽  
Nonsense Codon ◽  
Codon Context ◽  
Nonsense Codons

The effect of the 3' codon context on the efficiency of nonsense suppression in mammalian tissue culture cells has been tested. Measurements were made following the transfection of cells with a pRSVgal reporter vector that contained the classical Escherichia coli lacZ UAG allele YA559. The position of this mutation was mapped by virtue of its fortuitous creation of a CTAG MaeI restriction enzyme site. Determination of the local DNA sequence revealed a C-->T mutation at codon 600 of the lacZ gene: CAG-->TAG. Site-directed mutagenesis was used to create a series of vectors in which the base 3' to the nonsense codon was either A, C, G, or U. Suppression of the amber-containing reporter was achieved by cotransfection with genes for human tRNA(Ser) or tRNA(Gln) UAG nonsense suppressors and by growth in the translational error-promoting aminoglycoside drug G418. Nonsense suppression was studied in the human cell lines 293 and MRC5V1 and the simian line COS-7. Overall, the rank order for the effect of changes to the base 3' to UAG was C < G = U < A. This study confirms and extends earlier findings that in mammalian cells 3' C supports efficient nonsense suppression while 3' A is unsympathetic for read-through at nonsense codons. The rules for the mammalian codon context effect on nonsense suppression are therefore demonstrably different from those in E. coli.

Nonsense mutations in the human β-globin gene lead to unexpected levels of cytoplasmic mRNA accumulation

Blood ◽  
2000 ◽  
Vol 96 (8) ◽  
pp. 2895-2901 ◽  
Author(s):  
Luı́sa Romão ◽  
Ângela Inácio ◽  
Susana Santos ◽  
Madalena Ávila ◽  
Paula Faustino ◽  
...  
Keyword(s):  
Globin Gene ◽  
Mrna Level ◽  
Erythroid Differentiation ◽  
Mrna Levels ◽  
Globin Genes ◽  
Mrna Accumulation ◽  
Nonsense Mutations ◽  
Differentiation Induction ◽  
Nonsense Codons ◽  
Murine Erythroleukemia

Abstract Generally, nonsense codons 50 bp or more upstream of the 3′-most intron of the human β-globin gene reduce mRNA abundance. In contrast, dominantly inherited β-thalassemia is frequently associated with nonsense mutations in the last exon. In this work, murine erythroleukemia (MEL) cells were stably transfected with human β-globin genes mutated within each of the 3 exons, namely at codons 15 (TGG→TGA), 39 (C→T), or 127 (C→T). Primer extension analysis after erythroid differentiation induction showed codon 127 (C→T) mRNA accumulated in the cytoplasm at approximately 20% of the normal mRNA level. Codon 39 (C→T) mutation did not result in significant mRNA accumulation. Unexpectedly, codon 15 (TGG→TGA) mRNA accumulated at approximately 90%. Concordant results were obtained when reticulocyte mRNA from 2 carriers for this mutation was studied. High mRNA accumulation of codon 15 nonsense-mutated gene was revealed to be independent of the type of nonsense mutation and the genomic background in which this mutation occurs. To investigate the effects of other nonsense mutations located in the first exon on the mRNA level, nonsense mutations at codons 5, 17, and 26 were also cloned and stably transfected into MEL cells. After erythroid differentiation induction, mRNAs with a mutation at codon 5 or 17 were detected at high levels, whereas the mutation at codon 26 led to low mRNA levels. These findings suggest that nonsense-mediated mRNA decay is not exclusively dependent on the localization of mutations relative to the 3′-most intron. Other factors may also contribute to determine the cytoplasmic nonsense-mutated mRNA level in erythroid cells.

Sign in / Sign up

Export Citation Format

Share Document