Vulnerable exons, like ACADM exon 5, are highly dependent on maintaining a correct balance between splicing enhancers and silencers

Abstract

It is now widely accepted that aberrant splicing of constitutive exons is often caused by mutations affecting cis‐acting splicing regulatory elements, but there is a misconception that all exons have an equal dependency on splicing regulatory elements and thus a similar susceptibility to aberrant splicing. We investigated exonic mutations in ACADM exon 5 to experimentally examine their effect on splicing and found that 7 out of 11 tested mutations affected exon inclusion, demonstrating that this constitutive exon is particularly vulnerable to exonic splicing mutations. Employing ACADM exon 5 and 6 as models, we demonstrate that the balance between splicing enhancers and silencers, flanking intron length, and flanking splice site strength are important factors that determine exon definition and splicing efficiency of the exon in question. Our study shows that two constitutive exons in ACADM have different inherent vulnerabilities to exonic splicing mutations. This suggests that in silico prediction of potential pathogenic effects on splicing from exonic mutations may be improved by also considering the inherent vulnerability of the exon. Moreover, we show that single nucleotide polymorphism that affect either of two different exonic splicing silencers, located far apart in exon 5, all protect against both immediately flanking and more distant exonic splicing mutations.

Comprehensive splicing analysis of the alternatively spliced CHEK2 exons 8 and 10 reveals three enhancer/silencer‐rich regions and 38 spliceogenic variants

“…Understanding the regulatory patterns that control alternative splicing can provide valuable insights into disease aetiology and aid the development of ad hoc therapeutic strategies for splicing-related diseases [43,44]. However, exon recognition is a highly complex process that depends on multiple activators and repressors through a combinatorial control [27,45,46] of multiple factors:…”

“…Hence, the effect on splicing of SRE variants tends to be associated with partial/weak anomalous outcomes [47]. Nevertheless, an SRE variant can induce complete splicing anomalies, such as the SPINK5 variant c.891C>T, which shows simultaneous ESE (Tra2β) weakening and ESS (hnRNPA1) strengthening [49], or the ACADM gene, with a fine ESE/ESS balance (SRSF1, SRSF6/hnRNPA1) involved in exon 5 definition, where ESS variants counteract the splicing impact of ESE variants [46]. Both cases exemplify the complexity of the splicing process and may help to explain the still high inaccuracy of in silico predictions.…”

“…Hybrid minigenes are simple versatile tools to initially assess the impact of any sequence variation on splicing, as we have previously shown in the main BC susceptibility genes [19][20][21][22][23][24]31,49]. In this regard, the maintenance of the genomic context, including intron length, is essential for exon recognition and, therefore, for minigene design [25,27,46]. Even though the construct mgChk2_ex6-10 contains shortened introns due to their large sizes (1,849-6,439 bp), it generates the expected full-length transcript and mimics the prevalent alternative splicing events Δ(E8) and Δ(E10) of the endogenous CHEK2 gene [28,31].…”

Antisense Oligonucleotide Rescue of Deep-Intronic Variants Activating Pseudoexons in the 6-Pyruvoyl-Tetrahydropterin Synthase Gene

“…Surface plasmon resonance imaging (SPRi) was carried out as previously described [ 32 ]. Briefly, the biotinylated RNA-oligonucleotides described above were immobilized onto a G-strep sensorchip (SSENS) for 20 min.…”

Candidate genes and sequence variants for susceptibility to mycobacterial infection identified by whole-exome sequencing

“…SPRi was carried out as previously described ( Holm et al, 2022 ). Briefly, biotinylated RNA-oligonucleotides (GBP2-WT: 5′-UAUUUGGUGUUCUACGUCAUGA-teg-bio and GBP2-MUT: 5′-UAUUUGGUGUCCUACGUCAUGA-teg-bio) were immobilized onto a G-strep sensorchip (SSENS) for 20 min.…”