Variants outside donor and acceptor splice site motifs and impact on mRNA splicing
Precursor mRNAs are transcription products of human genes composed of exons interspersed with introns. Exon-intron boundaries are defined by multiple sequence motifs (Figure 1). Among these are the donor (5’) and acceptor (3’) splice site motifs based on the definitions by Burge et al (Burge, Tuschi, & Sharp, 1999): 11 bases for the donor splice site motif (from the 3 last exonic to the 8 first intronic bases); and 14 bases for the acceptor splice site motif (from the 12 last intronic to the first 2 exonic bases). The other significant motifs are BP sites and the polypyrimidine tract upstream of the 3’ splice site (Z. Wang & Burge, 2008). In mature mRNAs, introns are spliced out and exons are ligated by a complex cellular machinery called the spliceosome, containing small nuclear RNAs and proteins (Z. Wang & Burge, 2008). However, precursor mRNAs can undergo alternative splicing, leading to different mature mRNAs. This process is regulated by cisregulatory elements including splicing enhancers and silencers that recruit various RNA-binding proteins (Z. Wang & Burge, 2008). Exonic splicing regulatory elements (SREs) include exonic splicing enhancers (ESEs) and exonic splicing silencers (ESSs). Sequence variants that alter the composition, affinity, and function of spliceosomes can lead to the improper identification of exon-intron boundaries, thereby generating mRNAs that encode a premature termination codon, or otherwise encode a dysfunctional protein (G.-S. Wang & Cooper, 2007).
Variants within the first two bases or last three bases of the exon can alter the native splice sites and inactivate them. Exonic variants that introduce sequences that are identical or closely similar to donor or acceptor splice site sequences can be spliceogenic if the de novosplice site motif has sufficient activity to outcompete the native splice site. Exonic variants can also induce exon skipping through ESE loss and/or ESS gain (Cartegni, Hastings, Calarco, de Stanchina, & Krainer, 2006).
Intronic variants that abrogate BP sites, commonly located within the -18 to -44 nucleotide window (Signal, Gloss, Dinger, & Mercer, 2018), can lead to exon skipping (Khan et al., 2004; Wappenschmidt et al., 2012; K. Zhang, Nowak, Rushlow, Gallie, & Lohmann, 2008), intron retention (M. Li & Pritchard, 2000), or usage of new distant 3’ splice sites (Crotti et al., 2009). Deeper intronic variants, typically more than 100 nucleotides from exon-intron junctions, can lead to insertion of cryptic exons into the mature mRNA transcript by creating a new (or enhancing use of a cryptic) donor or acceptor motif, or by interfering with SREs [reviewed in (Vaz-Drago, Custódio, & Carmo-Fonseca, 2017)].