Figure 1.

The spf/ash ornithine transcarbamylase (OTC) mutation can be efficiently rescued by U1snRNA variants. (A) Schematic representation of the mouse OTC genomic sequence cloned as minigene in the pTB vector. Exonic and intronic sequences are represented by boxes and lines, respectively. The sequences, with exonic and intronic nucleotides in upper and lower cases, respectively, report (i) the authentic 5′ss (position +1 within intron), and (ii) the intronic cryptic 5′ss (positions +49). The nucleotide change (G > A) leading to the spf/ash phenotype is indicated. The schematic representation of engineered U1 and U7 snRNAs, with relative binding sites, is reported. Primers used for RT-PCR are indicated by arrows. (B) Evaluation of mouse OTC alternative splicing patterns in Hepa1-6 cells transiently transfected with wild-type or mutated minigenes alone or in combination with 1.5× molar excess of U1snRNA variants or the engineered U7snRNA. Amplified products were separated on 2% agarose gel (M, 100 bp molecular weight marker). Transcripts were validated by sequencing, whose electropherograms are reported below. The green arrow indicates the c.386G > A mutation.