Mol Cell Biol 16:4426-2235 (1996)

The Effect of Nonsense Mutations on Nuclear and Cytoplasmic Adenine Phosphoribosyltransferase RNA

Kessler, O. & Chasin, L.A.

We have analyzed Chinese hamster ovary (CHO) cell mutants bearing nonsense codons in 4 of the 5 exons of the adenine phosphoribosyltransferase (aprt) gene and have found a pattern of mRNA reduction similar to that seen in systems studied previously: a decrease in steady-state 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 aprt 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 aprt mRNA was found to be associated with polysomes, ruling out a model in which stable residual mRNA escapes degradation by avoiding translation initiation. A tetracycline-responsive form of the aprt gene was used to compare the stability of nonsense-containing and wild-type aprt mRNA without globally inhibiting transcription. In contrast to measurements made in the presence of actinomycin D, after inhibition of aprt transcription with tetracycline, a nonsense-mediated destabilization of aprt mRNA was indeed demonstrable. The increased rate of decay of cytoplasmic aprt 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 translation of translatability before it exits that compartment. Quantitation of the steady-state levels of transcripts containing introns revealed no accumulation of partially spliced aprt RNA, and thus no indication of nonsense-mediated aberrancies in splicing. Our results are consistent with a model in which translation facilitates the export of mRNA through a nuclear pore. However, the mechanism of this intriguing nucleo-cytoplasmic communication remains to be determined.