Analysis of a splice acceptor site mutation which produces multiple splicing abnormalities in the human...

The cloned argininosuccinate synthetase gene from a citrullinemia patient's fibroblast cell line revealed a single base substitution (G to C) within the splice acceptor site of the last intron. The mutation abolished normal RNA splicing, and, by cDNA analysis, three abnormal splicing pathways were demonstrated. The major pathway involved the activation of a cryptic acceptor site in the last exon that resulted in a deletion of seven nucleotides in the mature RNA. Another pathway involved a downstream cryptic acceptor site, that is 388 nucleotides downstream from the first cryptic site. Northern blot analysis showed that this second cryptic site is present on the minor 2.7-kilobase mRNA, but not on the major species of argininosuccinate synthetase mRNA, which is 1.7-kilobases in length. Using this aberrant cDNA as a probe, the cDNA of the 2.7-kilobase mRNA was isolated and studied. Sequence analysis suggests that this species of RNA is the one that bypasses the polyadenylation signal employed by the 1.7-kilobase RNA. Since both transcripts encounter the same translation termination codon, both RNAs should encode identical protein. Furthermore, a tract of 22 repeats of d(CA).(GT) is found at the 3' end of the gene and this repeat sequence is present on the 2.7-kilobase RNA. The third pathway of the abnormal splicing revealed a rare class of transcript that has the last intron retained in the mature RNA. This study shows that in human the intron inclusion can occur through a naturally occurring point mutation. All these abnormally spliced RNAs resulted in a protein reading frame shift.