Fig. 2: Results of functional analysis of the PP insertion.

a Results of the blood mRNA analysis demonstrated the presence of an additional GLB1 mRNA isoform with inclusion of the 18 bp fragment of the GLB1 intron 5 in patient and his mother, which is the heterozygous carrier of the PP insertion at the DNA level. b Fragment of the patient’s mutated β-GAL with the insertion of six amino acids (the 3D model was created by SWISS-MODEL based on the PDB:3WEZ template), located in the close proximity to the protein’s active site and two amino acids upstream of the catalytic residue Asn187. Catalytic residues are colored in pink, insertion is red and N8V ligand is blue. c The scheme of the GLB1 intron 5 fragment at the pre-mRNA level. The cryptic 18 bp exon (Ex. cr.) is located 36 bp upstream of the PP insertion (NPM1 cds) and was supposed to be activated by the PP-derived splicing enhancer motifs (E1-3). Splicing enhancers are enriched in binding sites of serine-rich proteins (SR), which promote the inclusion of the exons by interacting with spliceosome. d Location of studied ASMOs. Three clusters of splicing enhancer motifs were predicted by HExoSplice and were chosen as targets for ASMOs together with the cryptic exon (additional information is in Supplementary Figs. 1 and 2). e Fragment analysis and representative polyacrylamide gel electrophoresis visualization of PCR products obtained from minigene experiments. HEK293T cells were transfected with minigenes containing the fragment of the WT intron 5 and the mutated one (with PP insertion) alone (columns 2 and 3) or co-transfected with the mutated minigene and plasmids expressing modU7snRNAs (columns 4–17). Columns 4–8 (U7.1-5) correspond to modU7snRNAs targeting cryptic exon. Columns 9–13 (U7.S1–S5) correspond to the same modU7snRNAs but tailed with the splicing silencer hnRNPA1 motif. Columns 14–17 (U7.E1-E3) correspond to the modU7snRNAs targeting PP-derived splicing enhancer motifs. Error bars represent the standard deviation of biological replicates. The uncropped blots are presented in Supplementary Fig. 3. All blots derive from the same experiment and were processed in parallel.