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.

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.

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.

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.

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-mediated mRNA decay

2008 ◽  
Vol 36 (3) ◽  
pp. 514-516 ◽  
Author(s):  
Jikai Wen ◽  
Saverio Brogna
Keyword(s):  
Mammalian Cells ◽  
Mrna Decay ◽  
Translation Termination ◽  
Mrna Splicing ◽  
Current Understanding ◽  
Coupled Processes ◽  
Recent Developments ◽  
Nonsense Mediated Mrna Decay ◽  
Premature Translation Termination

Translation and mRNA decay are coupled processes; the link is most obvious in the case of NMD (nonsense-mediated mRNA decay). NMD is a mechanism that drastically reduces the level of mRNA harbouring PTCs (premature translation termination codons). The defining event in NMD is premature translation termination and the key question is: what distinguishes premature from normal translation termination? Surprisingly, in mammalian cells, PTC recognition is linked to pre-mRNA splicing. Here, we review the current understanding in view of recent developments.

scholarly journals Nonsense-Mediated mRNA Decay Effectors Are Essential for Zebrafish Embryonic Development and Survival

2009 ◽  
Vol 29 (13) ◽  
pp. 3517-3528 ◽  
Author(s):  
Nadine Wittkopp ◽  
Eric Huntzinger ◽  
Catrin Weiler ◽  
Jérôme Saulière ◽  
Steffen Schmidt ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Mrna Decay ◽  
Cultured Cells ◽  
Translation Termination ◽  
Zebrafish Genome ◽  
Zebrafish Embryos ◽  
Nonsense Mediated Mrna Decay ◽  
Nonsense Codons ◽  
Zebrafish Embryogenesis ◽  
Premature Translation Termination

ABSTRACT The nonsense-mediated mRNA decay (NMD) pathway promotes rapid degradation of mRNAs containing premature translation termination codons (PTCs or nonsense codons), preventing accumulation of potentially detrimental truncated proteins. In metazoa, seven genes (upf1, upf2, upf3, smg1, smg5, smg6, and smg7) have been identified as essential for NMD; here we show that the zebrafish genome encodes orthologs of upf1, upf2, smg1, and smg5 to smg7 and two upf3 paralogs. We also show that Upf1 is required for degradation of PTC-containing mRNAs in zebrafish embryos. Moreover, its depletion has a severe impact on embryonic development, early patterning, and viability. Similar phenotypes are observed in Upf2-, Smg5-, or Smg6-depleted embryos, suggesting that zebrafish embryogenesis requires an active NMD pathway. Using cultured cells, we demonstrate that the ability of a PTC to trigger NMD is strongly stimulated by downstream exon-exon boundaries. Thus, as in mammals and plants but in contrast to invertebrates and fungi, NMD is coupled to splicing in zebrafish. Our results together with previous studies show that NMD effectors are essential for vertebrate embryogenesis and suggest that the coupling of splicing and NMD has been maintained in vertebrates but lost in fungi and invertebrates.

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 mRNA Decay during Herpesvirus Infections: Interaction between a Putative Viral Nuclease and a Cellular Translation Factor

2001 ◽  
Vol 75 (21) ◽  
pp. 10272-10280 ◽  
Author(s):  
Pinghui Feng ◽  
David N. Everly ◽  
G. Sullivan Read
Keyword(s):  
Translation Initiation ◽  
Mammalian Cells ◽  
Mrna Decay ◽  
Point Mutations ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Viral Mrnas ◽  
Cellular Translation ◽  
Simplex Virus

ABSTRACT During lytic infections, the virion host shutoff (Vhs) protein (UL41) of herpes simplex virus destabilizes both host and viral mRNAs. By accelerating mRNA decay, it helps determine the levels and kinetics of viral and cellular gene expression. In vivo, Vhs shows a strong preference for mRNAs, as opposed to non-mRNAs, and degrades the 5′ end of mRNAs prior to the 3′ end. In contrast, partially purified Vhs is not restricted to mRNAs and causes cleavage of target RNAs at various sites throughout the molecule. To explain this discrepancy, we searched for cellular proteins that interact with Vhs using theSaccharomyces cerevisiae two-hybrid system. Vhs was found to interact with the human translation initiation factor, eIF4H. This interaction was verified by glutathioneS-transferase pull-down experiments and by coimmunoprecipitation of Vhs and epitope-tagged eIF4H from extracts of mammalian cells. The interaction was abolished by several point mutations in Vhs that abrogate its ability to degrade mRNAs in vivo. The results suggest that Vhs is a viral mRNA degradation factor that is targeted to mRNAs, and to regions of translation initiation, through an interaction with eIF4H.

scholarly journals Novel Upf2p Orthologues Suggest a Functional Link between Translation Initiation and Nonsense Surveillance Complexes

2000 ◽  
Vol 20 (23) ◽  
pp. 8944-8957 ◽  
Author(s):  
Joshua T. Mendell ◽  
Susan M. Medghalchi ◽  
Ross G. Lake ◽  
Erick N. Noensie ◽  
Harry C. Dietz
Keyword(s):  
Translation Initiation ◽  
Mammalian Cells ◽  
Translation Termination ◽  
Direct Interaction ◽  
Initiation Factor ◽  
Nuclear Targeting ◽  
Functional Link ◽  
Functional Conservation ◽  
Steady State Levels ◽  
Eukaryotic Initiation Factor 4G

ABSTRACT Transcripts harboring premature signals for translation termination are recognized and rapidly degraded by eukaryotic cells through a pathway known as nonsense-mediated mRNA decay (NMD). In addition to protecting cells by preventing the translation of potentially deleterious truncated peptides, studies have suggested that NMD plays a broader role in the regulation of the steady-state levels of physiologic transcripts. In Saccharomyces cerevisiae, three trans-acting factors (Upf1p to Upf3p) are required for NMD. Orthologues of Upf1p have been identified in numerous species, showing that the NMD machinery, at least in part, is conserved through evolution. In this study, we demonstrate additional functional conservation of the NMD pathway through the identification of Upf2p homologues inSchizosaccharomyces pombe and humans (rent2). Disruption ofS. pombe UPF2 established that this gene is required for NMD in fission yeast. rent2 was demonstrated to interact directly with rent1, a known trans-effector of NMD in mammalian cells. Additionally, fragments of rent2 were shown to possess nuclear targeting activity, although the native protein localizes to the cytoplasmic compartment. Finally, novel functional domains of Upf2p and rent2 with homology to eukaryotic initiation factor 4G (eIF4G) and other translational regulatory proteins were identified. Directed mutations within these so-called eIF4G homology (4GH) domains were sufficient to abolish the function of S. pombe Upf2p. Furthermore, using the two-hybrid system, we obtained evidence for direct interaction between rent2 and human eIF4AI and Sui1, both components of the translation initiation complex. Based on these findings, a novel model in which Upf2p and rent2 effects decreased translation and accelerated decay of nonsense transcripts through competitive interactions with eIF4G-binding partners is proposed.

Sign in / Sign up

Export Citation Format

Share Document