Nonsense mutations in the dihydrofolate reductase gene affect RNA processing.

Abstract

Steady-state dihydrofolate reductase (dhfr) mRNA levels were decreased as a result of nonsense mutations in the dhfr gene. Thirteen DHFR-deficient mutants were isolated after treatment of Chinese hamster ovary cells with UV irradiation. The positions of most point mutations were localized by RNA heteroduplex mapping, the mutated regions were isolated by cloning or by enzymatic amplification, and base changes were determined by DNA sequencing. Two of the mutants suffered large deletions that spanned the entire dhfr gene. The remaining 11 mutations consisted of nine single-base substitutions, one double-base substitution, and one single-base insertion. All of the single-base substitutions took place at the 3' position of a pyrimidine dinucleotide, supporting the idea that UV mutagenesis proceeds through the formation of pyrimidine dimers in mammalian cells. Of the 11 point mutations, 10 resulted in nonsense codons, either directly or by a frameshift, suggesting that the selection method favored a null phenotype. Biol., in press) showed that translation termination mutations in any of the internal exons of the gene gave rise to a low-RNA phenotype, whereas missense mutations in these exons or terminations in exon 6 (the final exon) did not affect dhfr mRNA levels. Nuclear run-on experiments showed that transcription of the mutant genes was normal. The stability of mature dhfr mRNA also was not affected, since (i) decay rates were the same in wild-type and mutant cells after inhibition of RNA synthesis with actinomycin D and (ii) intronless minigene versions of cloned wild-type and nonsense mutant genes were expressed equally after stable transfection. We conclude that RNA processing has been affected by these nonsense mutations and present a model in which both splicing and nuclear transport of an RNA molecule are coupled to its translation. Curiously, the low-RNA mutant phenotype was not exhibited after transfer of the mutant genes, suggesting that the transcripts of transfected genes may be processed differently than are those of their endogenous counterparts.In an effort to identify and understand those aspects of gene structure that play a role in gene expression in mammalian cells, we have been carrying out a detailed mutational analysis of the dihydrofolate reductase (dhfir) gene in Chinese hamster ovary (CHO) cells. dlifr exhibits many typical characteristics of mammalian housekeeping genes: it is 25 kilobase pairs (kbp) in size, contains five variously sized introns, and has a promoter region with no TATA box but with a very high G+C content and an Spl binding site (10,22,40,53). The basic strategy has been to isolate mutants that are deficient in DHFR enzyme activity; such mutants can be readily selected from a CHO cell line that is hemizygous at this locus (58, 59). To focus on lesions that may affect transcription or RNA processing, mutants can be screened for alterations that have affected dimfi-mRNA, either qualitatively or quantitatively.We have previously described spontaneous point mutations in th...

mRNA destabilization triggered by premature translational termination depends on at least three cis-acting sequence elements and one trans-acting factor.

“…In both prokaryotes and eukaryotes nonsense mutations in a gene can reduce the abundance of the mRNA transcribed from that gene (Morse and Yanofsky 1969;Losson and Lacroute 1979;Maquat et al 1981;Pelsy and Lacroute 1984;Baumann et al 1985;Nilsson et al 1987;Daar and Maquat 1988;Urlaub et al 1989;Cheng et al 1990;Gozalbo and Hohmann 1990;Barker and Beamon 1991;Gaspar et al 1991;Leeds et al 1991;Baserga and Benz 1992;Lim et al 1992;Cheng and Maquat 1993). Results of this study and others indicate that in S. cerevisiae this nonsense-mediated effect can be attributed to cytoplasmic events that are concurrent with mRNA translation.…”

Retinoblastoma protein associates with SP1 and activates the hamster dihydrofolate reductase promoter

“…Stables transfections of CHO K1 cells with eukaryotic expression vector for Rb (pCMVRb, Qin et al, 1992), or the empty vector, were performed by the calcium phosphate method (Urlaub et al, 1989). After 24 h expression, cells were selected with Geneticin (800 mg/ml) for 3 weeks.…”

Identification of a novel exonic mutation at -13 from 5' splice site causing exon skipping in a girl with mitochondrial acetoacetyl-coenzyme A thiolase deficiency.

“…In fibroblasts from patient GKO7, mRNA with the Q272STOP was prematurely terminated, although mRNA with exon 8 skipping did retain the original reading frame. Premature termination itself causes a marked reduction in the amount of mRNA, except when the termination codon is located in the penultimate or the last exons (30,31). Since exon 8 is not the penultimate or the last exon, premRNA with Q272STOP mutation was probably unstable, was degraded rapidly, and was undetectable.…”

“…4) (27); RT2, rat T2 (28); HT2, human T2 (11); RT1, rat mitochondrial 3-ketoacyl-CoA thiolase (29); RPT, rat peroxisomal 3-ketoacyl-CoA thiolase (30); and HPT, human peroxisomal 3-ketoacyl-CoA thiolase (31). These sequences were aligned to achieve maximum homologies.…”