Detection of Arabidopsis thaliana AtRAD1 cDNA variants and assessment of function by expression in a yeast rad1 mutant

The <i>Saccharomyces cerevisiae RAD1</i> and human <i>XPF</i> genes encode a subunit of a nucleotide excision repair endonuclease that also is implicated in some forms of homologous recombination. An <i>Arabidopsis thaliana</i> gene (<i>AtRAD1</i>) encoding the orthologous plant protein has been identified recently. Here we report the isolation of three structurally distinct <i>AtRAD1</i> cDNAs from <i>A. thaliana</i> leaf tissue RNA. One of the isolates (<i>AtRAD1-1</i>) corresponds to the cDNA previously shown to encode the full-length AtRad1 protein, whereas the other two (<i>AtRAD1-2, AtRAD1-3</i>) differ slightly in size due to variations at the 5′ end of exon 6 or the 3′ end of exon 7, respectively. The sequence differences argue that these cDNAs were probably templated by mRNAs generated via alternative splicing. Diagnostic polymerase chain reaction pointed to the presence of the <i>AtRAD1-1</i> and <i>AtRAD1-2</i> but not <i>AtRAD1-3</i> transcripts in bud and root tissue, and to a fourth transcript (<i>AtRAD1-4</i>), having both alterations identified in <i>AtRAD1-2</i> and <i>AtRAD1-3</i>, in root tissue. However, the low frequency of detection of <i>AtRAD1-3</i> and <i>AtRAD1-4</i> makes the significance of these tissue-specific patterns unclear. The predicted <i>AtRad1-2</i>, <i>AtRad1-3</i> and <i>AtRad1-4</i> proteins lack part of the region likely required for endonuclease complex formation. Expression of <i>AtRAD1-2</i> and <i>AtRAD1-3</i> in a yeast rad1 mutant did not complement the sensitivity to ultraviolet radiation or the recombination defect associated with the rad1 mutation. These results suggest that alternative splicing may modulate the levels of functional AtRad1 protein.<br />