Correction of aberrant splicing by modified U7 snRNAs.

(A) Correction of splicing of β-globin pre-mRNA by modified snRNAs. Boxes indicate exons; lines, introns; short bars above and below RNA, primers used in PCR and RT-PCR analysis. The dashed lines represent correct and aberrant splicing pathways. The modified U7 snRNA targeted to the 623 sequence (U7.623, see “Materials and methods”) is depicted under the pre-mRNA. (B) Structure of modified U7 snRNA constructs. Wild-type U7 snRNA includes a stem-loop structure, the U7-specific Sm sequence, and a sequence antisense to the 3′ end of histone pre-mRNA. The promoter and 3′ terminator regions are indicated. In modified U7 snRNAs, the Sm and antisense sequences were replaced with the spliceosomal Sm sequence SmOPT55 and with antisense sequences targeted to the β-globin pre-mRNA (see “Materials and methods”). The SmOPT site is boxed and the antisense sequences are underlined. (C) Splicing modification antisense assay. Cells lines were created stably expressing the enhanced green fluorescence (EGFP) gene in which the coding sequence was interrupted by the IVS2-654 or IVS2-705U β-globin intron 2 (see “Results”). Proper splicing and translation of EGFP-654 or EGFP-705U elicited by modified antisense U7 snRNA and visualized by either fluorescence microscopy (middle panels; magnification × 4) or flow cytometry (bottom panels) provide a positive readout for antisense activity. In this and subsequent figures histograms plot EGFP fluorescence intensity versus cell number.