Abnormal mRNA splicing resulting from K201N. (A) RT-PCR of a full-length STAT1α, STAT1β, STAT1 fragment running from exon 7 to exon 10, and glyceraldehyde-3-phosphate dehydrogenase from mRNA extracted from the EBV-B cells of a healthy control (WT/WT), P1, and persons with the following genotypes: K201N/WT, P696S/P696S, and 1928insA/1928insA (−/−). This result is representative of 3 independent experiments. H₂O was used as a negative control for PCR. (B) Schematic diagram of the STAT1α and STAT1β mRNA in the cells of P1. The upper band corresponds to the form with an MW identical to that of the WT mRNA, with normal splicing and containing the K201N mutation (red line). The lower band was observed for the cells of P1 and corresponds to forms of the STAT1α and STAT1β mRNAs lacking exon 8. The exons are numbered with Roman numerals. (C) Schematic diagram of the plasmid used for exon trapping. The genomic STAT1 region from nucleotides 15844 to 16416 (NC_000002) was inserted into the pSPL3 plasmid, between the XhoI and BamHI sites, with or without the K201N mutation (line in red). Exon 8 is shown in the green box and exon 9 in the blue box. SD6 and SA2 primer positions are indicated. (D) HEK293T and COS-7 cells were transfected with no vector (−), pSPL3 mock vector (M), pSPL3 vector containing the WT STAT1 gene (WT), and pSPL3 vector containing the K201N-mutated STAT1 gene (K201N). RT-PCR was carried out to amplify the splicing products 24 hours after transfection. (Top panel) PCR with the SD6 and SA2 primers. (Bottom panel) PCR with the glyceraldehyde-3-phosphate dehydrogenase primers. This result is representative of 2 independent experiments. (E) Schematic diagram of the 3 forms of mRNA splicing products: (i) exon 8 and exon 9 plus vector sequence; (ii) exon 9 plus vector sequence; and (iii) 263-bp product corresponding to exonic sequence in the vector.