Abstract
Noncanonical splice-site mutations are an important cause of inherited diseases. Based on in vitro and stem-cell-based studies, some splice-site variants show a stronger splice defect than expected based on their predicted effects, suggesting that other sequence motifs influence the outcome. We investigated whether splice defects due to human-inherited-disease-associated variants in noncanonical splice-site sequences in ABCA4, DMD, and TMC1 could be rescued by strengthening the splice site on the other side of the exon. Noncanonical 5′- and 3′-splice-site variants were selected. Rescue variants were introduced based on an increase in predicted splice-site strength, and the effects of these variants were analyzed using in vitro splice assays in HEK293T cells. Exon skipping due to five variants in noncanonical splice sites of exons in ABCA4, DMD, and TMC1 could be partially or completely rescued by increasing the predicted strengths of the other splice site of the same exon. We named this mechanism “splicing interdependency”, and it is likely based on exon recognition by splicing machinery. Awareness of this interdependency is of importance in the classification of noncanonical splice-site variants associated with disease and may open new opportunities for treatments.
Highlights
Pre-mRNA splicing of multi-exon genes is performed by the spliceosome, which recognizes specific sequence motifs at exon boundaries [1,2,3]
Decreased splicing scores often result in exon skipping, but exon elongation and partial exon truncation are observed when alternative splice sites are recognized by the splicing machinery [6,7,8]
When present alone in a minigene splice construct and transfected into Human Embryonic Kidney 293T (HEK293T) cells, it resulted in different Exon 39/40 skipping RNA products (Figure 1)
Summary
Pre-mRNA splicing of multi-exon genes is performed by the spliceosome, which recognizes specific sequence motifs at exon boundaries [1,2,3]. Variants in these motifs can change the binding strength of the spliceosome, which can alter splicing [4]. This can result in disease when necessary (parts of) exons are skipped or (parts of) introns are retained, causing frameshifts, premature stop codons, or disruptions in protein folding [5]. The above-mentioned splicing algorithms assess the strengths of the 5 and 3 splice sites independently, not taking into account that exon recognition may depend on the strength of the splice site at the other side of an exon
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