International Journal of Molecular Sciences Article In or Out? New Insights on Exon Recognition through Splice-Site Interdependency 1,2, 1,2, 1,3,4 1 Mubeen Khan y, Stéphanie S. Cornelis y , Riccardo Sangermano , Iris J.M. Post , 1 1 1,4 1,2, Amber Janssen Groesbeek , Jan Amsu , Christian Gilissen , Alejandro Garanto z , 1,2, 1,2, Rob W.J. Collin z and Frans P.M. Cremers * 1 Department of Human Genetics, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands; [email protected] (M.K.); [email protected] (S.S.C.); [email protected] (R.S.); [email protected] (I.J.M.P.); [email protected] (A.J.G.); [email protected] (J.A.); [email protected] (C.G.); [email protected] (A.G.); [email protected] (R.W.J.C.) 2 Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands 3 Ocular Genomics Institute, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114, USA 4 Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands * Correspondence: [email protected]; Tel.: +31-24-3614017; Fax: +31-24-3668752 These authors contributed equally to this work. y These authors contributed equally to this work. z Received: 7 February 2020; Accepted: 23 March 2020; Published: 26 March 2020 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 50- and 30-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. Keywords: Pre-mRNA; splicing; 50 and 30 splice sites; interdependency 1. Introduction Pre-mRNA splicing of multi-exon genes is performed by the spliceosome, which recognizes specific sequence motifs at exon boundaries [1–3]. 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]. Int. J. Mol. Sci. 2020, 21, 2300; doi:10.3390/ijms21072300 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 2300 2 of 13 In order to predict the effect of genetic variants on splicing, many splice-site prediction programs have been created. Five splicing prediction programs are used in the commonly used program Alamut Visual (Interactive Biosoftware, Rouen, France, version 2.7) to increase the reliability of this in silico analysis. Splicing prediction scores are provided for both the reference and alternative sequence, not only for the splice sites, indicating the known exon–intron boundaries but also alternative (cryptic) splice sites in nearby sequences. Decreased splicing scores often result in exon skipping, but exon elongation and partial exon truncation are also observed when alternative splice sites are recognized by the splicing machinery [6–8]. Different algorithms are used in various splicing prediction programs. SpliceSiteFinder-like [9,10], MaxEntScan [11], NNSPLICE [12], GeneSplicer [13], and Human Splicing Finder [14] are incorporated into the Alamut software and take into account a maximum of 80 nucleotides on either side of the variant. The exons in human DNA are relatively small (average 145–150 bp; mean 120 bp) [15,16] and introns are generally much larger (average 5.5 kb; mean 3.3 kb) [17,18], which likely forms the basis for an initial “exon recognition” by the spliceosome. Surprisingly, the above-mentioned splicing algorithms assess the strengths of the 50 and 30 splice sites independently, not taking into account that exon recognition may also depend on the strength of the splice site at the other side of an exon. In 2002, Hefferon et al. showed how exon skipping due to a short polypyrimidine tract upstream of CFTR Exon 9 at the 30 splice site could be rescued by an adenine insertion between positions +3 and +4, strengthening the 50 splice site of Exon 9. In vivo studies of mice and sheep supported this finding [19]. Later it was found that natural skipping of Exon 6 of the CHRNE gene could be reduced by improving either the 50 or 30 splice site of Exon 6 by changing at least four nucleotides in the sequence [20], indicating that if one of the splice sites of CHRNE Exon 6 is weak and the other is strong, normal splicing can still take place. By studying the effect of a predicted very mild 30-splice-site variant (c.5461-10T>C) in ABCA4 (NM_000350.2) in individuals with Stargardt disease (STGD1), we observed a very strong splice defect (Exon 39 or Exon 39 and 40 skipping) in mRNA isolated from patient-derived photoreceptor precursor cells [21]. We hypothesize that the large effect of the very mild 30-splice-site variant was due to a weak 50 splice site of Exon 39. Here, we aimed to study whether splice-site interdependency represents a more general phenomenon. Next to the noncanonical splice-site (NCSS) variant c.5461-10T>C in ABCA4, we selected additional predicted weak 30 NCSS variants in introns of DMD (NM_004006.2) and TMC1 (NM_138691.2), which were accompanied by relatively weak 50 splice sites [22,23]. Each of them has been previously shown to result in exon skipping and was associated with inherited human disease. Employing in vitro minigene splice assays to strengthen the 50 splice site by changing their sequence towards the 50 NCSS consensus sequence [24] enabled us to study the effect on splicing of the concerned exon as well as adjacent exons. Similarly, we investigated whether the exon skipping of two ABCA4 exons due to NCSS variants at the 50 splice site could be corrected by strengthening the 30 splice site, to investigate whether the dependency between splice sites works both ways. 2. Results 2.1. Rescuing the Exon-Skipping Effect of 30-Splice-Site Variants The 30-splice-site variant c.5461-10T>C constitutes the most frequent severe ABCA4 variant in STGD1. 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 (Figure1). Densitometric analysis showed that RNA transcribed from the wild-type (WT) construct also showed a fair amount of exon skipping (1% of Exon 39 skipping and 31% of Exon 39–40 skipping, Figure1C, Supplementary Table S1), also shown previously [21]. The BA26 minigene construct carrying c.5461-10T>C was also mutagenized to contain the alternative nucleotides A, C, or T at Position c.5584+4. Based on Human Splicing Finder (HSF), the +4A variant strengthened this 5’ splice site Int. J. Mol. Sci. 2020, 21, 2300 3 of 13 significantly (Figure1E; from 83.8 to 92.1). The +4C and +4T variants, also according to HSF, did not strengthen this splice site, but were inserted to investigate another hypothesis. The +4 to +6 nucleotides were G’GT, which were part of a weak predicted 50 splice site (HSF: 69.0) which could negatively influence proper Exon 39 recognition. The +4G>C and +4G>T changes could potentially remove this effect and thereby result in less Exon 39 skipping in the presence of the WT 30-splice-site sequence c.5461-10T, or the mutant variant c.5461-10C. The c.5584+4G>A variant, but not c.5584+4G>C or c.5584+4G>T, clearly rescued the splicing defect due to c.5461-10T>C (Figure1D–F, Supplementary Figure S1). Interestingly, the c.[5461-10T>C;5584+G>A] construct showed more correctly spliced mRNA than the WT construct. However, compared to the WT mRNA, a new band of ~400 nucleotides was present too, but this band could not be sequenced, which suggested that it was probably a heteroduplex fragment. As shown in Figure1A,D, the +4G>C and +4G>T changes did not result in a clear rescue of erroneous splicing in the presence of the WT or mutant 30 splice site, respectively, suggesting that the G’GT sequence at Positions +4 to +6 did not significantly affect the strength of the 50 splice site. Thereby, we showed that exon skipping due to a weak variant in the 30 splice site of ABCA4 Exon 39 could be fully rescued by strengthening the 50 splice site of the same exon. The next variant we investigated for splice-site interdependency was the pathogenic NCSS mutation c.1705-5T>G at the 30 splice site of DMD Exon 15, which is associated with Becker muscular dystrophy (www.lovd.nl/DMD)[23]. Minigenes of DMD Exons 14–16 were used to examine the effect of this mutation and a putative rescuing variant c.1812+4T>A on the splicing of these exons.