Summary: | The lecithin:cholesterol acyltransferase (LCAT) gene encodes a plasma enzyme
that plays a key role in the metabolism of high-density lipoproteins (HDL). Previous
mutations associated with LCAT deficiency syndromes have been identified in the
coding regions of the LCAT gene. Recently, our laboratory has found an intron mutation
in three patients with a form of LCAT deficiency previously described as fish-eye
disease (FED). The in vitro expression of the intron mutant has been shown to result in
the intron retention. Since the natural mutation occurs in a putative branchpoint
consensus sequence, we hypothesized that the point mutation might disrupt the splicing
of the LCAT pre-mRNA.
To test the hypothesis, two other novel mutations, i.e., LCAT IVS4-MUT-1 (T->G)
and MUT-2 (T->A), were introduced into the same site of the natural mutation
(IVS4:T->C22),). After stable transfection of the mutated LCAT minigenes into BHK
cells, neither LCAT activity nor LCAT protein could be detected in the culture medium of
the IVS4-MUT-1 and MUT-2 cell lines, as was previously described for the natural
mutation. To determine the effects of the introduced mutations on the splicing of pre-
mRNA, total RNA from transfected BHK cells was used for RT-PCR analysis. All BHK
cell lines were shown to transcribe the integrated LCAT minigenes. However, the sizes
of these LCAT messages indicated that intron 4 was retained in the IVS4-MUT-1 and
MUT-2 cell lines. Subsequent sequence analysis of the RT-PCR products demonstrated
that the unspliced intronic sequences contained the introduced mutations, suggesting
that the observed retention of intron 4 of the LCAT gene is the result of the specific loss
of a thymine residue two bases upstream of the branchpoint adenosine.
In attempts to investigate the possible mechanisms responsible for the defective
splicing and to study further the functional significance of the branchpoint sequence, a
series of mutations was generated in the whole region of the branchpoint sequence.
After these intron mutants were transiently expressed in HEK-293 cells, the efficiency of
pre-mRNA splicing was analyzed using RT-PCR as well as the measurement of LCAT
activity. The results revealed that (1) the mutation of the branchpoint adenosine to any
other nucleotide completely abolished the splicing; (2) the insertion of a normal branch
site into the intronic sequence of the natural (IVS4-22c) or the branchpoint (IVS4-20t)
mutant restored normal splicing; (3) the natural mutation could be partially suppressed
by changing its consensus sequence from CCCCGAC to CCCCAAC; and (4) other
single-base changes, especially around the branchpoint adenosine residue, significantly
decreased 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) resulted in 2.7-fold increase in splicing efficiency.
These results have demonstrated that the branchpoint sequence, although only
weakly conserved in mammals, can be of essential importance for accurate and efficient
splicing of human nuclear pre-mRNA and have contributed to better understanding of
the mechanism of branch-site selection during pre-mRNA splicing. The findings also
suggest that a DNA polymorphism involving the branchpoint sequence of an intron
might affect the efficiency of RNA splicing and thus have significant clinical implications.
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