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

Abstract

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 H. In this communication, we show by using genetics and Si nuclease transcript mapping that the premature termination of 1-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 13-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 13-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. mRNA exists to be translated into protein. However, evidence suggests that in the process of being translated, the abundance of an mRNA can be altered. For some mRNAs, abundance is increased if the coding region is not translated in full. Examples of this type include mRNAs for the replication-dependent human histones (10,18,26); human c-fos (19,41,42); murine c-myc (50); yeast MATel (35); and hamster, chicken, murine, and sea urchin P-tubulin (16,17,34,51,52). In each case, translation to at least a certain point within the coding region of the mRNA appears to be required for mRNA degradation, and nonsense codons that reside upstream of that point are associated with an increase in mRNA abundance. While similar in this regard, however, each of these mRNAs is not degraded as a function of the same features of translation. Histone mRNA degradation at the end of S phase requires that ribosomes translate to within a specific distance of a hairpin structure that resides in the 3' untranslated region. In contrast, c-fos, c-myc, and MATod mRNA degradation seems to require ribosome translation across a specific region of the coding sequence. And, as another variation, ,B-tubulin mRNA decay depends upon an excess of free tubulin that presumably interacts with the first 4 amino acids of an actively elongating, nascent peptide. (2,...

mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor.

“…In both prokaryotes and eukaryotes nonsense mutations in a gene can reduce the abundance of the mRNA transcribed from that gene (Morse and Yanofsky 1969;Losson and Lacroute 1979;Maquat et al 1981;Pelsy and Lacroute 1984;Baumann et al 1985;Nilsson et al 1987;Daar and Maquat 1988;Urlaub et al 1989;Cheng et al 1990;Gozalbo and Hohmann 1990;Barker and Beamon 1991;Gaspar et al 1991;Leeds et al 1991;Baserga and Benz 1992;Lim et al 1992;Cheng and Maquat 1993). Results of this study and others indicate that in S. cerevisiae this nonsense-mediated effect can be attributed to cytoplasmic events that are concurrent with mRNA translation.…”

The APC gene product in normal and tumor cells.

“…All of the truncated forms that were not detected were predicted to be smaller than 80 kDa. Other studies have indicated that truncating nonsense mutations can lead to decreased mRNA abundance (26). Also, some abnormal RB tumor suppressor gene transcripts predicted to exist in the lymphoid cells of patients with hereditary retinoblastoma could not be identified (27), raising the possibility that very premature termination of APC mRNA translation could lead to decreased7 mRNA stability.…”

Expression of the laminin‐A chain is down‐regulated by a non‐canonical polyadenylation signal

“…However, no introns in 3' UTR (downstream of stop codons) have been found in natural, nonpathological mRNA. In those cases where an intron occurs in the 3' UTR because of premature stop codons in the coding region (Lim et al, 1992), the resulting mRNA seems to be labile, leading to reduced expression. In our constructs, the situation is still different, as the coding region of the reporter gene has not been altered.…”