Secondary structure prediction of pre-mRNA sequence. Secondary structure of the wt pre-mRNA sequence (A) and the mutated pre-mRNA sequence (B) predicted by mfold. Many of the 5′ donor splice site residues (5 residues: −1U, +1G, +2U, +3A, and +7C) that bind to U1 snRNA were observed to be free in the wt sequence but prebound or physically constrained in a double-stranded form in the mutated sequence. The regions corresponding to residues that bind to U1 snRNA are marked by the lavender-shaded area. The mutated residue locations are also marked by the magenta-shaded area and identified by a thick arrow. The inset images in each panel correspond to a close-up view of the region of pre-mRNA that binds to U1 snRNA in the 3-dimensional model of the wt and mutated structures generated from mfold secondary structure prediction on the RNAComposer Web server. The model structure is depicted in stick format (light green). The residues that bind to U1 snRNA are shown in blue. Hydrogen bonds are depicted as magenta dots.