title: Effects of nonsense mutations on nuclear and cytoplasmic adenine phosphoribosyltransferase RNA.
abstract: We have analyzed Chinese hamster ovary (CHO) cell mutants bearing nonsense codons in four of the five 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 is 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 mRNAs 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 or 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 hence 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 nucleocytoplasmic communication remains to be determined.
authors: Department of Biological Sciences, Columbia University, New York, New York 10027, USA.
authors: O
authors: O
authors: Kessler
authors: L A
authors: LA
authors: Chasin
mesh_terms: D000228:Adenine Phosphoribosyltransferase / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D009694:Nucleic Acid Precursors / Q000378:metabolism; D010443:Peptide Chain Termination, Translational; D017354:Point Mutation; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D009694:Nucleic Acid Precursors; D012333:RNA, Messenger; D000228:Adenine Phosphoribosyltransferase