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. 2019 Apr 22;47(11):5922–5935. doi: 10.1093/nar/gkz259

Figure 1.

Figure 1.

Positional preferences in splice site selection. (A) Schematic representation of the LMNA exon 11 splicing event. Wild-type and mutant (HGPS) splicing events are represented by dotted black lines. Grey box in exon 11 represents the 150 nt removed in the LMNA C1824T mutant. (B) Splice site strength calculation for the normal 5′ SS, unmutated alternative 5′ SS (WT) and the mutated alternative 5′SS (MUT) using the Maximum Entropy Model (MAXENT), Maximum Dependence Decomposition Model (MDD), Markov Model (MM) or Weight Matrix Model (WMM) and Splice site score. (C) Design of mutant minigene constructs to compare the effect of splice site sequences at the proximal and distal locations relative to intron 11/12. Splice sites with the same color have the same sequence. Blue: WT, alternative splice site; yellow: WT, normal splice site; green: mutant, alternative splice site. (D, F) RT-PCR analysis for LMNA/Δ150 expression of each mutant. 293T cells were transfected with 1 μg of the indicated plasmids. RNA was harvested at 72 h post transfection. DsRed expression served as an internal loading control. (E, G) Quantification of mRNA isoforms expressed as relative usage of the normal vs. alternative 5′ SS. Data are expressed as mean ± s.d. from three independent experiments. WT: LMNA wild-type minigene construct; MUT: LMNA C1824U mutant minigene construct; 2X-NORM: minigene construct with normal splice site sequence at both proximal and distal splice site positions; MUT/2X-ALT: minigene construct with C1824U mutant alternative splice site sequence at both proximal and distal splice site positions; WT/2X-ALT: minigene construct with un-mutated alternative splice sequence at both proximal and distal splice positions; SWAP: minigene construct with C1824U mutant alternative splice site sequence at the proximal splice site and normal splice site sequence at the distal splice site; WT/NORM-ALT MUT: minigene construct with C1824U mutant alternative splice site sequence at the proximal splice site and unmutated alternative splice site sequence at the distal splice site; NC: negative control (PCR reaction mix without cDNA template).