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

We have previously identified a point mutation (intervening sequence (TVS) 4: T --> C) in the branchpoint consensus sequence of intron 4 of the lecith in:cholesterol acyltransferase (LCAT) gene in patients with fish-eye diseas e. To investigate the possible mechanisms responsible for the defective spl icing, we made a series of mutations in the branchpoint sequence and expres sed these mutants in HEK-293 cells followed by the analysis of pre-mRNA spl icing 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 in sertion 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 nucle otide change (G --> A) within the branchpoint consensus sequence; and 4) ot her single base changes, particularly around the branchpoint adenosine resi due, significantly decrease the efficiency of splicing and thus enzyme acti vity. Surprisingly, the nucleotide transversion at the last position of the branchpoint sequence (i.e. IVS4-25a or -25g) results in a 2.7-fold increas e in splicing efficiency. Therefore, these observations clearly establish t he functional significance of the branchpoint sequence of intron 4 for the splicing of the human LCAT mRNA precursors.