Characterization of the effects of mutations in the putative branchpoint sequence of intron 4 on the splicing within the human lecithin:cholesterol acyltransferase gene

We have previously identified a point mutation (intervening sequence (TVS) 4: T --> C) in the branchpoint consensus sequence of intron 4 of the lecithin:cholesterol acyltransferase (LCAT) gene in patients with fish-eye disease. To investigate the possible mechanisms responsible for the defective splicing, we made a series of mutations in the branchpoint sequence and expressed these mutants in HEK-293 cells followed by the analysis of pre-mRNA splicing using reverse transcriptase-polymerase chain reaction as well as LCAT activity assay. The results reveal that 1) the mutation of the branchpoint adenosine to any other nucleotide completely abolishes splicing; 2) the insertion of a normal branch site into the intronic sequence of the natural (IVS4-22c) or the branchpoint (IVS4-20t) mutant completely restores splicing; 3) the natural mutation can be partially rescued by making a single nucleotide change (G --> A) within the branchpoint consensus sequence; and 4) other single base changes, particularly around the branchpoint adenosine residue, significantly decrease the efficiency of splicing and thus enzyme activity. Surprisingly, the nucleotide transversion at the last position of the branchpoint sequence (i.e. IVS4-25a or -25g) results in a 2.7-fold increase in splicing efficiency. Therefore, these observations clearly establish the functional significance of the branchpoint sequence of intron 4 for the splicing of the human LCAT mRNA precursors.