Nonsense codons in human beta-globin mRNA result in the production of mRNA degradation products

Human beta zero-thalassemic beta-globin genes harboring either a frameshift or a nonsense mutation that results in the premature termination of beta-globin mRNA translation have been previously introduced into the germ line of mice (S.-K. Lim, J.J. Mullins, C.-M. Chen, K. Gross, and L.E. Maquat, EMBO J. 8:2613-2619, 1989). Each transgene produces properly processed albeit abnormally unstable mRNA as well as several smaller RNAs in erythroid cells. These smaller RNAs are detected only in the cytoplasm and, relative to mRNA, are longer-lived and are missing sequences from either exon I or exons I and II. In this communication, we show by using genetics and S1 nuclease transcript mapping that the premature termination of beta-globin mRNA translation is mechanistically required for the abnormal RNA metabolism. We also provide evidence that generation of the smaller RNAs is a cytoplasmic process: the 5' ends of intron 1-containing pre-mRNAs were normal, the rates of removal of introns 1 and 2 were normal, and studies inhibiting RNA synthesis with actinomycin D demonstrated a precursor-product relationship between full-length mRNA and the smaller RNAs. In vivo, about 50% of the full-length species that undergo decay are degraded to the smaller RNAs and the rest are degraded to undetectable products. Exposure of erythroid cells that expressed a normal human beta-globin transgene to either cycloheximide or puromycin did not result in the generation of the smaller RNAs. Therefore, a drug-induced reduction in cellular protein synthesis does not reproduce this aspect of cytoplasmic mRNA metabolism. These data suggest that the premature termination of beta-globin mRNA translation in either exon I or exon II results in the cytoplasmic generation of discrete mRNA degradation products that are missing sequences from exon I or exons I and II. Since these degradation products appear to be the same for all nonsense codons tested, there is no correlation between the position of translation termination and the sites of nucleolytic cleavage.

[1]  K. Beemon,et al.  Nonsense codons within the Rous sarcoma virus gag gene decrease the stability of unspliced viral RNA. , 1991, Molecular and cellular biology.

[2]  M. Greenberg,et al.  Two distinct destabilizing elements in the c-fos message trigger deadenylation as a first step in rapid mRNA decay. , 1991, Genes & development.

[3]  R. Wisdom,et al.  The protein-coding region of c-myc mRNA contains a sequence that specifies rapid mRNA turnover and induction by protein synthesis inhibitors. , 1991, Genes & development.

[4]  R. Harland,et al.  Endonucleolytic cleavage of a maternal homeo box mRNA in Xenopus oocytes. , 1990, Genes & development.

[5]  L. Maquat,et al.  Translation to near the distal end of the penultimate exon is required for normal levels of spliced triosephosphate isomerase mRNA , 1990, Molecular and cellular biology.

[6]  R Parker,et al.  Identification and comparison of stable and unstable mRNAs in Saccharomyces cerevisiae , 1990, Molecular and cellular biology.

[7]  R. Parker,et al.  Translation and a 42-nucleotide segment within the coding region of the mRNA encoded by the MAT alpha 1 gene are involved in promoting rapid mRNA decay in yeast. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[8]  H. Hanafusa,et al.  Processing of 9E3 mRNA and regulation of its stability in normal and Rous sarcoma virus-transformed cells , 1989, Molecular and cellular biology.

[9]  D. Cleveland,et al.  Autoregulatory control of beta-tubulin mRNA stability is linked to translation elongation. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[10]  L. Chasin,et al.  Nonsense mutations in the dihydrofolate reductase gene affect RNA processing , 1989, Molecular and cellular biology.

[11]  G. Brawerman.  mRNA decay: Finding the right targets , 1989, Cell.

[12]  P. Sharp,et al.  5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits transcription elongation by RNA polymerase II in vitro. , 1989, The Journal of biological chemistry.

[13]  J. Belasco,et al.  Mechanisms of mRNA decay in bacteria: a perspective. , 1988, Gene.

[14]  S. R. Kushner,et al.  Stabilization of discrete mRNA breakdown products in ams pnp rnb multiple mutants of Escherichia coli K-12 , 1988, Journal of bacteriology.

[15]  H. Kazazian,et al.  New amber mutation in a beta-thalassemic gene with nonmeasurable levels of mutant messenger RNA in vivo. , 1988, The Journal of clinical investigation.

[16]  B. Brandhorst,et al.  Stabilization of tubulin mRNA by inhibition of protein synthesis in sea urchin embryos , 1988, Molecular and cellular biology.

[17]  S. Baserga,et al.  Nonsense mutations in the human beta-globin gene affect mRNA metabolism. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[18]  J. Pachter,et al.  Autoregulated changes in stability of polyribosome-bound beta-tubulin mRNAs are specified by the first 13 translated nucleotides , 1988, Molecular and cellular biology.

[19]  L. Maquat,et al.  Premature translation termination mediates triosephosphate isomerase mRNA degradation , 1988, Molecular and cellular biology.

[20]  J. Ross.  Messenger RNA turnover in eukaryotic cells. , 1988, Molecular biology & medicine.

[21]  J. Pachter,et al.  Autoregulation of tubulin expression is achieved through specific degradation of polysomal tubulin mRNAs , 1987, Cell.

[22]  R. Lührmann,et al.  A monoclonal antibody against 2,2,7-trimethylguanosine that reacts with intact, class U, small nuclear ribonucleoproteins as well as with 7-methylguanosine-capped RNAs. , 1987, European journal of biochemistry.

[23]  A. Skoultchi,et al.  Coupling of replication type histone mRNA levels to DNA synthesis requires the stem-loop sequence at the 3' end of the mRNA. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[24]  N. Heintz,et al.  Sequences controlling histone H4 mRNA abundance. , 1987, The EMBO journal.

[25]  R. Graves,et al.  Translation is required for regulation of histone mRNA degradation , 1987, Cell.

[26]  S. Peltz,et al.  Histone mRNA degradation in vivo: the first detectable step occurs at or near the 3' terminus , 1986, Molecular and cellular biology.

[27]  J. Ross,et al.  H4 histone messenger RNA decay in cell-free extracts initiates at or near the 3' terminus and proceeds 3' to 5'. , 1986, Journal of molecular biology.

[28]  M. Greenberg,et al.  Effect of protein synthesis inhibitors on growth factor activation of c-fos, c-myc, and actin gene transcription , 1986, Molecular and cellular biology.

[29]  T. Shenk,et al.  Sequence-independent autoregulation of the adenovirus type 5 E1A transcription unit , 1985, Molecular and cellular biology.

[30]  J. Lingrel,et al.  Erythroid‐specific expression of human beta‐globin genes in transgenic mice. , 1985, The EMBO journal.

[31]  M. Potash,et al.  Consequences of frameshift mutations at the immunoglobulin heavy chain locus of the mouse. , 1985, The EMBO journal.

[32]  K. Takeshita,et al.  Intranuclear defect in beta-globin mRNA accumulation due to a premature translation termination codon. , 1984, Blood.

[33]  L. Maquat,et al.  mRNA-deficint β°-thaladssemia results from a single nucleotide deletion , 1982 .

[34]  R. Weinmann,et al.  Mechanism of action of dichloro-beta-D-ribofuranosylbenzimidazole: effect on in vitro transcription. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[35]  R. Losson,et al.  Interference of nonsense mutations with eukaryotic messenger RNA stability. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[36]  H. Fan,et al.  Regulation of protein synthesis in mammalian cells. II. Inhibition of protein synthesis at the level of initiation during mitosis. , 1970, Journal of molecular biology.

[37]  B. Hogan.  he effect of inhibitors of protein synthesis on the level of ribosomal subunits in ascites cells. , 1969, Biochimica et biophysica acta.

[38]  C. Stanners.  The effect of cycloheximide on polyribosomes from hamster cells. , 1966, Biochemical and biophysical research communications.

[39]  R. Traut,et al.  THE PUROMYCIN REACTION AND ITS RELATION TO PROTEIN SYNTHESIS. , 1964, Journal of molecular biology.

[40]  S. Peltz,et al.  Regulation of mRNA turnover in eukaryotic cells. , 1991, Critical reviews in eukaryotic gene expression.

[41]  M. Greenberg,et al.  The c-fos transcript is targeted for rapid decay by two distinct mRNA degradation pathways. , 1989, Genes & development.

[42]  S. Pestka.  The use of inhibitors in studies on protein synthesis. , 1974, Methods in enzymology.