Figure 2.

Analysis of splicing regulatory elements (SRE) of SBDS exon 2 and rescue of c.258+2T>C mutation with engineered U1snRNAs. (A) Upper panel: sequences of SBDS exon 2 and surrounding introns are indicated in upper and lower case, respectively. The regions masked by the engineered U7snRNAs, indicated above the sequence, are underlined. The highly conserved dinucleotides of the exonic cryptic 3′ss and 5′ss are boxed and double underlined, respectively. The total number of exonic splicing enhancers (ESE) and exonic splicing silencers (ESS) is reported in the heat map. SRE: Splicing Regulatory Elements. Lower panel: Evaluation of SBDS splicing patterns in HEK293T cells co-transfected with sbds^wt or sbds⁺² minigenes alone or with engineered U7snRNAs. The schematic representation of transcripts is reported on the right; (B) Schematic representation of SBDS exon 2, in upper case, intron 2, in lower case, and of U1snRNA variants exploited to redirect the spliceosome to the mutated 5′ss. The splicing pattern of HEK293T cells transfected with wild-type or mutated minigenes alone or in combination with U1snRNAs is indicated below. The schematic representation of transcripts is reported on the right; and (C) Evaluation of SBDS splicing patterns in HEK293T cells co-transfected with the sbds⁺² minigene, U1¹snRNA, and U7snRNA variants. Amplified products were separated on 2.5% agarose gel. M, 100 bp molecular weight marker. The schematic representation of the transcripts (with exons not in scale) is reported on the right.