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Nonsense codons in human beta-globin mRNA result in the production of mRNA degradation products
S K Lim1, C D Sigmund, K W Gross
1Department of Human Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263.
Abstract:
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.
Insights
Premature termination of beta-globin mRNA translation leads to abnormal RNA metabolism. This process generates smaller cytoplasmic RNA fragments, independent of translation inhibition drugs.
Area of Science:
- Molecular Biology
- Genetics
- RNA Metabolism
Background:
- Beta-thalassemia involves mutations in beta-globin genes, leading to reduced or absent beta-globin production.
- Previous studies introduced human beta-thalassemic genes into mice, resulting in unstable mRNA and smaller RNA species in erythroid cells.
Purpose of the Study:
- To investigate the mechanistic link between premature mRNA translation termination and abnormal RNA metabolism.
- To determine if smaller RNA generation is a cytoplasmic process.
- To assess the role of cellular protein synthesis in this RNA degradation pathway.
Main Methods:
- Germline transmission of human beta-thalassemic beta-globin transgenes in mice.
- S1 nuclease transcript mapping and genetic analysis.
- Inhibition of RNA synthesis using actinomycin D.
- Treatment of erythroid cells with cycloheximide and puromycin.
Main Results:
- Premature translation termination is mechanistically required for abnormal RNA metabolism.
- Generation of smaller RNAs is a cytoplasmic process, independent of nuclear events.
- A precursor-product relationship exists between full-length mRNA and smaller RNAs.
- About 50% of decaying mRNA degrades into these smaller RNA species.
- Drug-induced reduction in protein synthesis does not replicate smaller RNA generation.
Conclusions:
- Premature termination of beta-globin mRNA translation in exon I or II triggers the cytoplasmic generation of discrete mRNA degradation products.
- These degradation products are missing sequences from exon I or exons I and II.
- The observed RNA degradation pattern is consistent across different nonsense codons, irrespective of termination site.