Mammalian nonsense codons can be cis effectors of nuclear mRNA half-life.

Abstract

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 shutof 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.Triosephosphate isomerase (TPI; EC 5.3.1.1) catalyzes the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate and has an important role in glycolysis, gluconeogenesis, fatty acid synthesis, and the pentose shunt (29). Frameshift and nonsense mutations within the human TPI gene that result in a nonsense codon within the first three-fourths of the translated portion of TPI mRNA reduce the abundance of TPI mRNA to 20 to 30% of normal (10,11). Since the reduction is characteristic of both nuclear and cytoplasmic cell fractions and is not attributable to a decrease in the half-life of cytoplasmic TPI mRNA, the degradative process has been rationalized to involve nuclear TPI RNA (10, 11). Such deductive reasoning presents a dilemma when one considers a plausible degradative mechanism because nonsense codons are known to be recognized only in the cytoplasm during the process of mRNA translation-a process that in mammalian cells is generally thought to be spatially and temporally distinct from the processes of nuclear RNA metabolism. Indeed, degradation is likely to be initiated by the premature termination of cytoplasmic mRNA translation, since the reduction in the abundance of a nonsense codoncontaining mRNA is abrogated by a hairpin structure in the 5' untranslated region, which acts in cis to inhibit translation in...

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