NONSENSE BUT NOT MISSENSE MUTATIONS CAN DECREASE THE ABUNDANCE OF NUCLEAR MESSENGER-RNA FOR THE MOUSE MAJOR URINARY PROTEIN, WHILE BOTH TYPES OF MUTATIONS CAN FACILITATE EXON SKIPPING

In an effort to understand the mechanisms by which nonsense codons aff ect RNA metabolism in mammalian cells, nonsense mutations were generat ed within the gene for the secretory major urinary protein (MUP) of mi ce. The translation of MUP mRNA normally begins within exon 1 and term inates within exon 6, the penultimate exon. Through the use of Norther n (RNA) blot hybridization and assays that couple reverse transcriptio n and PCR, a nonsense mutation within codon 50 of exon 2 or codon 143 of exon 5 was found to reduce the abundance of fully spliced, nuclear MUP mRNA to 10 to 20% of normal without an additional reduction in the abundance of cytoplasmic mRNA. In contrast, a nonsense mutation withi n codon 172 of exon 5 was found to have no effect on the abundance of MUP mRNA. These findings suggest that a boundary between nonsense muta tions that do and do net reduce the abundance of nuclear mRNA exists w ithin the exon preceding the exon that harbors the normal site of tran slation termination. In this way, the boundary is analogous to the bou ndary that exists within the penultimate exon of the human gene for th e cytosolic enzyme triosephosphate isomerase. Assays for exon skipping , i.e., the removal of an exon as a part of the flanking introns durin g the process of splicing, reveal that 0.1, 2.0, and 0.1% of MUP mRNA normally lack exon 5, exon 6, and exons 5 plus 6, respectively. Relati ve to normal, the two nonsense mutations within exon 5 increase the ab undance of RNA lacking exon 5 on average 20-fold and increase the abun dance of RNA lacking exons 5 plus 6 on average 5-fold. Since only one of these nonsense mutations also reduces the abundance of fully splice d nuclear mRNA to 10 to 20% of normal, the two mechanisms by which a n onsense mutation can alter nuclear RNA metabolism must be distinct. Th e analysis of missense mutations within codons 143 and 172, some of wh ich retain the nonsense mutation, indicates that the reduction in the abundance of fully spliced nuclear mRNA is dependent upon the prematur e termination of MUP mRNA translation, whereas skipping is attributabl e to nonsense mutation mediated changes in exon 5 structure rather tha n to the premature termination of translation. The increase in exon 5 skipping by either the nonsense or missense mutations within codon 172 correlates with a decrease in the complementarity of exon 5 to U1 snR NA. This suggests that a 5' splice site may extend as far as 12 nucleo tides into the upstream exon, which is, to our knowledge, the largest extension.