We have previously identified distinct nuclear and mitochondrial isoforms of dUTPase in human cells, reporting the cDNA sequence of the nuclear isoform (DUT-N). We now report a cDNA corresponding to the mitochondrial isoform (DUT-M). The DUT-M cDNA contains an 252-amino acid open reading frame, encoding a protein with a predicted M(r) of 26,704. The amino- terminal region of the protein contains an arginine-rich, 69-residue mitochondrial targeting presequence that is absent in the mature protein. In vitro transcription and translation of the DUT-M cDNA results in the production of a precursor protein with an apparent molecular mass of 31 kDa as judged by SDS-polyacrylamide gel electrophoresis. The DUT-M precursor is enzymatically active and immunoreacts with a dUTPase-specific monoclonal antibody. Mitochondrial import and processing studies demonstrate that the DUT-M precursor is processed into a 23-kDa protein and imported into mitochondria in vitro. Isoelectric focusing experiments demonstrate that the DUT-N has a pI of 6.0, while the processed form of DUT-M has a more basic pI of 8.1, measurements that are in agreement with predicted values. Studies aimed at understanding the expression of these isoforms were performed utilizing quiescent and replicating 34Lu human lung fibroblasts as a model cell culture system. Northern blot analysis, employing an isoform-specific probe, demonstrates that DUT-N and DUT-M are encoded by two distinct mRNA species of 1.1 and 1.4 kilobases, respectively. Western and Northern blot analysis reveal that DUT-M protein and mRNA are expressed in a constitutive fashion, independent of cell cycle phase or proliferation status. In contrast, DUT-N protein and mRNA levels are tightly regulated to coincide with nuclear DNA replication status. Because DUT-N and DUT-M have identical amino acid and cDNA sequences in their overlapping regions, we set out to determine if they were encoded by the same gene. The 5' region of the gene encoding dUTPase was isolated and characterized by a combination of Southern hybridization and DNA sequencing. These analyses demonstrate that the dUTPase isoforms are encoded by the same gene with isoform-specific transcripts arising through the use of alternative 5' exons. This finding represents the first example in humans of alternative 5' exon usage to generate differentially expressed nuclear and mitochondrial specific protein isoforms.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology