MAMMALIAN NONSENSE CODONS CAN BE CIS EFFECTORS OF NUCLEAR MESSENGER-RNA HALF-LIFE

Frameshift and nonsense mutations within the gene for human triosephos phate isomerase (TPI) that generate a nonsense codon within the first three-fourths of the protein coding region have been found to reduce t he abundance of the product mRNA that copurifies with nuclei. The cell ular process and location of the nonsense codon-mediated reduction hav e proven difficult to elucidate for technical reasons. We show here, u sing electron microscopy to judge the purity of isolated nuclei, that the previously established reduction to 25% of the normal mRNA level i s evident for nuclei that are free of detectable cytoplasmic contamina tion. 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 shutoff of TPI gene transcr iption upon the addition of serum to serum-deprived cells. Finally, th e finding that deletion of a 5' splice site of the TPI gene results pr edominantly but not exclusively in the removal by splicing (i.e., skip ping) 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 t he decay mechanism, indicate that nonsense codon recognition takes pla ce after splicing and triggers decay solely in cis. The possibility th at decay takes place during the process of mRNA export from the nucleu s to the cytoplasm is discussed.