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. 1998 Jun 15;17(12):3484–3494. doi: 10.1093/emboj/17.12.3484

Binary specification of nonsense codons by splicing and cytoplasmic translation.

R Thermann 1, G Neu-Yilik 1, A Deters 1, U Frede 1, K Wehr 1, C Hagemeier 1, M W Hentze 1, A E Kulozik 1
PMCID: PMC1170685  PMID: 9628884

Abstract

Premature translation termination codons resulting from nonsense or frameshift mutations are common causes of genetic disorders. Complications arising from the synthesis of C-terminally truncated polypeptides can be avoided by 'nonsense-mediated decay' of the mutant mRNAs. Premature termination codons in the beta-globin mRNA cause the common recessive form of beta-thalassemia when the affected mRNA is degraded, but the more severe dominant form when the mRNA escapes nonsense-mediated decay. We demonstrate that cells distinguish a premature termination codon within the beta-globin mRNA from the physiological translation termination codon by a two-step specification mechanism. According to the binary specification model proposed here, the positions of splice junctions are first tagged during splicing in the nucleus, defining a stop codon operationally as a premature termination codon by the presence of a 3' splicing tag. In the second step, cytoplasmic translation is required to validate the 3' splicing tag for decay of the mRNA. This model explains nonsense-mediated decay on the basis of conventional molecular mechanisms and allows us to propose a common principle for nonsense-mediated decay from yeast to man.

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Selected References

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  1. Aoufouchi S., Yélamos J., Milstein C. Nonsense mutations inhibit RNA splicing in a cell-free system: recognition of mutant codon is independent of protein synthesis. Cell. 1996 May 3;85(3):415–422. doi: 10.1016/s0092-8674(00)81119-8. [DOI] [PubMed] [Google Scholar]
  2. Applequist S. E., Selg M., Raman C., Jäck H. M. Cloning and characterization of HUPF1, a human homolog of the Saccharomyces cerevisiae nonsense mRNA-reducing UPF1 protein. Nucleic Acids Res. 1997 Feb 15;25(4):814–821. doi: 10.1093/nar/25.4.814. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baserga S. J., Benz E. J., Jr Beta-globin nonsense mutation: deficient accumulation of mRNA occurs despite normal cytoplasmic stability. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2935–2939. doi: 10.1073/pnas.89.7.2935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baserga S. J., Benz E. J., Jr Nonsense mutations in the human beta-globin gene affect mRNA metabolism. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2056–2060. doi: 10.1073/pnas.85.7.2056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baumann B., Potash M. J., Köhler G. Consequences of frameshift mutations at the immunoglobulin heavy chain locus of the mouse. EMBO J. 1985 Feb;4(2):351–359. doi: 10.1002/j.1460-2075.1985.tb03636.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Belgrader P., Cheng J., Maquat L. E. Evidence to implicate translation by ribosomes in the mechanism by which nonsense codons reduce the nuclear level of human triosephosphate isomerase mRNA. Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):482–486. doi: 10.1073/pnas.90.2.482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Belgrader P., Cheng J., Zhou X., Stephenson L. S., Maquat L. E. Mammalian nonsense codons can be cis effectors of nuclear mRNA half-life. Mol Cell Biol. 1994 Dec;14(12):8219–8228. doi: 10.1128/mcb.14.12.8219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Belgrader P., Maquat L. E. Nonsense but not missense mutations can decrease the abundance of nuclear mRNA for the mouse major urinary protein, while both types of mutations can facilitate exon skipping. Mol Cell Biol. 1994 Sep;14(9):6326–6336. doi: 10.1128/mcb.14.9.6326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carter M. S., Doskow J., Morris P., Li S., Nhim R. P., Sandstedt S., Wilkinson M. F. A regulatory mechanism that detects premature nonsense codons in T-cell receptor transcripts in vivo is reversed by protein synthesis inhibitors in vitro. J Biol Chem. 1995 Dec 1;270(48):28995–29003. doi: 10.1074/jbc.270.48.28995. [DOI] [PubMed] [Google Scholar]
  10. Carter M. S., Li S., Wilkinson M. F. A splicing-dependent regulatory mechanism that detects translation signals. EMBO J. 1996 Nov 1;15(21):5965–5975. [PMC free article] [PubMed] [Google Scholar]
  11. Cheng J., Belgrader P., Zhou X., Maquat L. E. Introns are cis effectors of the nonsense-codon-mediated reduction in nuclear mRNA abundance. Mol Cell Biol. 1994 Sep;14(9):6317–6325. doi: 10.1128/mcb.14.9.6317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cheng J., Fogel-Petrovic M., Maquat L. E. Translation to near the distal end of the penultimate exon is required for normal levels of spliced triosephosphate isomerase mRNA. Mol Cell Biol. 1990 Oct;10(10):5215–5225. doi: 10.1128/mcb.10.10.5215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cheng J., Maquat L. E. Nonsense codons can reduce the abundance of nuclear mRNA without affecting the abundance of pre-mRNA or the half-life of cytoplasmic mRNA. Mol Cell Biol. 1993 Mar;13(3):1892–1902. doi: 10.1128/mcb.13.3.1892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Daar I. O., Maquat L. E. Premature translation termination mediates triosephosphate isomerase mRNA degradation. Mol Cell Biol. 1988 Feb;8(2):802–813. doi: 10.1128/mcb.8.2.802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dietz H. C., Kendzior R. J., Jr Maintenance of an open reading frame as an additional level of scrutiny during splice site selection. Nat Genet. 1994 Oct;8(2):183–188. doi: 10.1038/ng1094-183. [DOI] [PubMed] [Google Scholar]
  16. Dietz H. C. Nonsense mutations and altered splice-site selection. Am J Hum Genet. 1997 Mar;60(3):729–730. [PMC free article] [PubMed] [Google Scholar]
  17. Dietz H. C., Valle D., Francomano C. A., Kendzior R. J., Jr, Pyeritz R. E., Cutting G. R. The skipping of constitutive exons in vivo induced by nonsense mutations. Science. 1993 Jan 29;259(5095):680–683. doi: 10.1126/science.8430317. [DOI] [PubMed] [Google Scholar]
  18. Enssle J., Kugler W., Hentze M. W., Kulozik A. E. Determination of mRNA fate by different RNA polymerase II promoters. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10091–10095. doi: 10.1073/pnas.90.21.10091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Goossen B., Hentze M. W. Position is the critical determinant for function of iron-responsive elements as translational regulators. Mol Cell Biol. 1992 May;12(5):1959–1966. doi: 10.1128/mcb.12.5.1959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gray N. K., Hentze M. W. Iron regulatory protein prevents binding of the 43S translation pre-initiation complex to ferritin and eALAS mRNAs. EMBO J. 1994 Aug 15;13(16):3882–3891. doi: 10.1002/j.1460-2075.1994.tb06699.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Green S., Issemann I., Sheer E. A versatile in vivo and in vitro eukaryotic expression vector for protein engineering. Nucleic Acids Res. 1988 Jan 11;16(1):369–369. doi: 10.1093/nar/16.1.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hagemeier C. Site-directed mutagenesis using a uracil-containing phagemid template. Methods Mol Biol. 1996;57:45–54. doi: 10.1385/0-89603-332-5:45. [DOI] [PubMed] [Google Scholar]
  23. Hall G. W., Thein S. Nonsense codon mutations in the terminal exon of the beta-globin gene are not associated with a reduction in beta-mRNA accumulation: a mechanism for the phenotype of dominant beta-thalassemia. Blood. 1994 Apr 15;83(8):2031–2037. [PubMed] [Google Scholar]
  24. Hentze M. W., Caughman S. W., Casey J. L., Koeller D. M., Rouault T. A., Harford J. B., Klausner R. D. A model for the structure and functions of iron-responsive elements. Gene. 1988 Dec 10;72(1-2):201–208. doi: 10.1016/0378-1119(88)90145-x. [DOI] [PubMed] [Google Scholar]
  25. Hentze M. W., Kühn L. C. Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8175–8182. doi: 10.1073/pnas.93.16.8175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hentze M. W., Rouault T. A., Caughman S. W., Dancis A., Harford J. B., Klausner R. D. A cis-acting element is necessary and sufficient for translational regulation of human ferritin expression in response to iron. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6730–6734. doi: 10.1073/pnas.84.19.6730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Humphries R. K., Ley T. J., Anagnou N. P., Baur A. W., Nienhuis A. W. Beta O-39 thalassemia gene: a premature termination codon causes beta-mRNA deficiency without affecting cytoplasmic beta-mRNA stability. Blood. 1984 Jul;64(1):23–32. [PubMed] [Google Scholar]
  28. Kessler O., Chasin L. A. Effects of nonsense mutations on nuclear and cytoplasmic adenine phosphoribosyltransferase RNA. Mol Cell Biol. 1996 Aug;16(8):4426–4435. doi: 10.1128/mcb.16.8.4426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Klausner R. D., Rouault T. A., Harford J. B. Regulating the fate of mRNA: the control of cellular iron metabolism. Cell. 1993 Jan 15;72(1):19–28. doi: 10.1016/0092-8674(93)90046-s. [DOI] [PubMed] [Google Scholar]
  30. Kozak M. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986 Jan 31;44(2):283–292. doi: 10.1016/0092-8674(86)90762-2. [DOI] [PubMed] [Google Scholar]
  31. Kugler W., Enssle J., Hentze M. W., Kulozik A. E. Nuclear degradation of nonsense mutated beta-globin mRNA: a post-transcriptional mechanism to protect heterozygotes from severe clinical manifestations of beta-thalassemia? Nucleic Acids Res. 1995 Feb 11;23(3):413–418. doi: 10.1093/nar/23.3.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Li S., Leonard D., Wilkinson M. F. T cell receptor (TCR) mini-gene mRNA expression regulated by nonsense codons: a nuclear-associated translation-like mechanism. J Exp Med. 1997 Mar 17;185(6):985–992. doi: 10.1084/jem.185.6.985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lim S. K., Maquat L. E. Human beta-globin mRNAs that harbor a nonsense codon are degraded in murine erythroid tissues to intermediates lacking regions of exon I or exons I and II that have a cap-like structure at the 5' termini. EMBO J. 1992 Sep;11(9):3271–3278. doi: 10.1002/j.1460-2075.1992.tb05405.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lim S. K., Sigmund C. D., Gross K. W., Maquat L. E. Nonsense codons in human beta-globin mRNA result in the production of mRNA degradation products. Mol Cell Biol. 1992 Mar;12(3):1149–1161. doi: 10.1128/mcb.12.3.1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Liu X., Mertz J. E. Sequence of the polypyrimidine tract of the 3'-terminal 3' splicing signal can affect intron-dependent pre-mRNA processing in vivo. Nucleic Acids Res. 1996 May 1;24(9):1765–1773. doi: 10.1093/nar/24.9.1765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Lozano F., Maertzdorf B., Pannell R., Milstein C. Low cytoplasmic mRNA levels of immunoglobulin kappa light chain genes containing nonsense codons correlate with inefficient splicing. EMBO J. 1994 Oct 3;13(19):4617–4622. doi: 10.1002/j.1460-2075.1994.tb06783.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Maquat L. E., Kinniburgh A. J., Rachmilewitz E. A., Ross J. Unstable beta-globin mRNA in mRNA-deficient beta o thalassemia. Cell. 1981 Dec;27(3 Pt 2):543–553. doi: 10.1016/0092-8674(81)90396-2. [DOI] [PubMed] [Google Scholar]
  38. Maquat L. E. When cells stop making sense: effects of nonsense codons on RNA metabolism in vertebrate cells. RNA. 1995 Jul;1(5):453–465. [PMC free article] [PubMed] [Google Scholar]
  39. Mashima Y., Murakami A., Weleber R. G., Kennaway N. G., Clarke L., Shiono T., Inana G. Nonsense-codon mutations of the ornithine aminotransferase gene with decreased levels of mutant mRNA in gyrate atrophy. Am J Hum Genet. 1992 Jul;51(1):81–91. [PMC free article] [PubMed] [Google Scholar]
  40. McCaughan K. K., Brown C. M., Dalphin M. E., Berry M. J., Tate W. P. Translational termination efficiency in mammals is influenced by the base following the stop codon. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5431–5435. doi: 10.1073/pnas.92.12.5431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Menon K. P., Neufeld E. F. Evidence for degradation of mRNA encoding alpha-L-iduronidase in Hurler fibroblasts with premature termination alleles. Cell Mol Biol (Noisy-le-grand) 1994 Nov;40(7):999–1005. [PubMed] [Google Scholar]
  42. Naeger L. K., Schoborg R. V., Zhao Q., Tullis G. E., Pintel D. J. Nonsense mutations inhibit splicing of MVM RNA in cis when they interrupt the reading frame of either exon of the final spliced product. Genes Dev. 1992 Jun;6(6):1107–1119. doi: 10.1101/gad.6.6.1107. [DOI] [PubMed] [Google Scholar]
  43. Pantopoulos K., Gray N. K., Hentze M. W. Differential regulation of two related RNA-binding proteins, iron regulatory protein (IRP) and IRPB. RNA. 1995 Apr;1(2):155–163. [PMC free article] [PubMed] [Google Scholar]
  44. Pantopoulos K., Hentze M. W. Nitric oxide signaling to iron-regulatory protein: direct control of ferritin mRNA translation and transferrin receptor mRNA stability in transfected fibroblasts. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1267–1271. doi: 10.1073/pnas.92.5.1267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pogulis R. J., Vallejo A. N., Pease L. R. In vitro recombination and mutagenesis by overlap extension PCR. Methods Mol Biol. 1996;57:167–176. doi: 10.1385/0-89603-332-5:167. [DOI] [PubMed] [Google Scholar]
  46. Pulak R., Anderson P. mRNA surveillance by the Caenorhabditis elegans smg genes. Genes Dev. 1993 Oct;7(10):1885–1897. doi: 10.1101/gad.7.10.1885. [DOI] [PubMed] [Google Scholar]
  47. Qian L., Theodor L., Carter M., Vu M. N., Sasaki A. W., Wilkinson M. F. T cell receptor-beta mRNA splicing: regulation of unusual splicing intermediates. Mol Cell Biol. 1993 Mar;13(3):1686–1696. doi: 10.1128/mcb.13.3.1686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Ruiz-Echevarria M. J., Czaplinski K., Peltz S. W. Making sense of nonsense in yeast. Trends Biochem Sci. 1996 Nov;21(11):433–438. doi: 10.1016/s0968-0004(96)10055-4. [DOI] [PubMed] [Google Scholar]
  49. Ruiz-Echevarría M. J., González C. I., Peltz S. W. Identifying the right stop: determining how the surveillance complex recognizes and degrades an aberrant mRNA. EMBO J. 1998 Jan 15;17(2):575–589. doi: 10.1093/emboj/17.2.575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Simpson S. B., Stoltzfus C. M. Frameshift mutations in the v-src gene of avian sarcoma virus act in cis to specifically reduce v-src mRNA levels. Mol Cell Biol. 1994 Mar;14(3):1835–1844. doi: 10.1128/mcb.14.3.1835. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Takeshita K., Forget B. G., Scarpa A., Benz E. J., Jr Intranuclear defect in beta-globin mRNA accumulation due to a premature translation termination codon. Blood. 1984 Jul;64(1):13–22. [PubMed] [Google Scholar]
  52. Thein S. L., Hesketh C., Taylor P., Temperley I. J., Hutchinson R. M., Old J. M., Wood W. G., Clegg J. B., Weatherall D. J. Molecular basis for dominantly inherited inclusion body beta-thalassemia. Proc Natl Acad Sci U S A. 1990 May;87(10):3924–3928. doi: 10.1073/pnas.87.10.3924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Urlaub G., Mitchell P. J., Ciudad C. J., Chasin L. A. Nonsense mutations in the dihydrofolate reductase gene affect RNA processing. Mol Cell Biol. 1989 Jul;9(7):2868–2880. doi: 10.1128/mcb.9.7.2868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Vetter B., Schwarz C., Kohne E., Kulozik A. E. Beta-thalassaemia in the immigrant and non-immigrant German populations. Br J Haematol. 1997 May;97(2):266–272. doi: 10.1046/j.1365-2141.1997.342674.x. [DOI] [PubMed] [Google Scholar]
  55. Visa N., Izaurralde E., Ferreira J., Daneholt B., Mattaj I. W. A nuclear cap-binding complex binds Balbiani ring pre-mRNA cotranscriptionally and accompanies the ribonucleoprotein particle during nuclear export. J Cell Biol. 1996 Apr;133(1):5–14. doi: 10.1083/jcb.133.1.5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Zhang J., Maquat L. E. Evidence that translation reinitiation abrogates nonsense-mediated mRNA decay in mammalian cells. EMBO J. 1997 Feb 17;16(4):826–833. doi: 10.1093/emboj/16.4.826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zillmann M., Zapp M. L., Berget S. M. Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles. Mol Cell Biol. 1988 Feb;8(2):814–821. doi: 10.1128/mcb.8.2.814. [DOI] [PMC free article] [PubMed] [Google Scholar]

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