Abstract
A translationally silent single nucleotide mutation in exon 44 (E44) of the von Willebrand factor (VWF) gene is associated with inefficient removal of intron 44 in a von Willebrand disease (VWD) patient. This intron retention (IR) event was previously attributed to reordered E44 secondary structure that sequesters the normal splice donor site. We propose an alternative mechanism: the mutation introduces a cryptic splice donor site that interferes with the function of the annotated site to favor IR. We evaluated both models using minigene splicing reporters engineered to vary in secondary structure and/or cryptic splice site content. Analysis of splicing efficiency in transfected K562 cells suggested that the mutation-generated cryptic splice site in E44 was sufficient to induce substantial IR. Mutations predicted to vary secondary structure at the annotated site also had modest effects on IR and shifted the balance of residual splicing between the cryptic site and annotated site, supporting competition among the sites. Further studies demonstrated that introduction of cryptic splice donor motifs at other positions in E44 did not promote IR, indicating that interference with the annotated site is context dependent. We conclude that mutant deep exon splice sites can interfere with proper splicing by inducing IR.
Highlights
Aberrant splicing is a common cause of human genetic disease with various studies estimating that 15–60% of disease-causing mutations disrupt pre-mRNA splicing ([1,2]; reviewed in [3])
The current study examined an alternative model for intron retention in this von Willebrand factor (VWF) patient, based on the observation that the mutation generates a strong 5 splice site motif deep within exon 44 (E44)
Weak 5 splice sites at exon/intron borders are often susceptible to regulation, and there is precedence for the ability of cryptic exon splice site motifs to promote downstream intron retention [34]
Summary
Aberrant splicing is a common cause of human genetic disease with various studies estimating that 15–60% of disease-causing mutations disrupt pre-mRNA splicing ([1,2]; reviewed in [3]). 5–20% of cancer-predisposing mutations adversely affect splicing [4]. Mutations can create or destroy splice site motifs, alter enhancer or silencer elements that promote or inhibit recognition of splice sites, or alter the function of splicing regulatory proteins ([5,6,7,8] and references therein). Even synonymous single nucleotide exonic mutations that do not impact known regulatory motifs can adversely affect pre-mRNA splicing patterns. Distinguishing the mechanisms by which such mutations disrupt splicing requires detailed studies that will improve therapeutic strategies
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