EFFECTS OF NONSENSE MUTATIONS ON NUCLEAR AND CYTOPLASMIC ADENINE PHOSPHORIBOSYLTRANSFERASE RNA

We have analyzed Chinese hamster ovary (CHO) cell mutants bearing nons ense codons in four of the five exons of the adenine phosphoribosyltra nsferase (aprt) gene and have found a pattern of mRNA reduction simila r to that seen in systems studied previously: a decrease in steady-sta te mRNA levels of 5- to 10-fold for mutations in exons 1, 2, and 4 but little effect for mutations in the 3'-most exon (exon 5). Nuclear apr t mRNA levels showed a similar decrease. Nonsense-containing aprt mRNA decayed at the same rate as wild-type mRNA in these cell lines after inhibition of transcription with actinomycin D. Nonsense-containing ap rt mRNA is associated with polysomes, ruling out a model in which stab le residual mRNA escapes degradation by avoiding translation initiatio n. A tetracycline-responsive form of the aprt gene was used to compare the stability of nonsense-containing and wild-type aprt mRNAs without globally inhibiting transcription. In contrast to measurements made i n the presence of actinomycin D, after inhibition of aprt transcriptio n with tetracycline, a nonsense-mediated destabilization of aprt mRNA was indeed demonstrable. The increased rate of decay of cytoplasmic ap rt mRNA seen here could account for the nonsense-mediated reduction in steady-state levels of aprt mRNA. However, the low levels of nonsense -bearing aprt mRNA in the nucleus suggest a sensibility of mRNA to tra nslation or translatability before it exits that compartment. Quantita tion of the steady-state levels of transcripts containing introns reve aled no accumulation of partially spliced aprt RNA and hence no indica tion of nonsense-mediated aberrancies in splicing. Our results are con sistent with a model in which translation facilitates the export of mR NA through a nuclear pore. However, the mechanism of this intriguing n ucleocytoplasmic communication remains to be determined.