(A) Top: Western blot of RAD50 protein (left) and RT-PCR of RAD50 transcript (right) from control and patient fibroblasts. Bottom: Intron 21 with c.3390-1119_3390-1115del deletion (blue) and downstream poison exon (italics underlined). (B–G) Splicing analysis in HEK293 cells (see Supplemental Methods for additional information). (B) RAD50 minigene constructs confirming exon inclusion for the Δ5 mutant. (C) Effects of deletions and substitutions within 5′-TGAGT-3′ on minigene splicing. (D) Exon inclusion with short versus long PPTs. Data are shown as the mean ± SD. (E) Role of pyrimidine-rich sequences for minigene splicing. (F) Overexpression of U2AF promotes aberrant splicing. (G) In vitro binding of U2AF to RNA oligonucleotides spanning the Δ5 region. (H) Model for a role of distal extension of the poly-pyrimidine region in promoting exon inclusion through U2AF accumulation, if devoid of an AG. (I) Correction of RAD50 splicing, protein level, and ionizing radiation–induced ATM signaling by AMO treatment.