Effects on in vitro splicing of 2′-O-methyl antisense oligonucleotides against U6atac and U4atac. (A) Schematic of U6atac (top strand) and U4atac (bottom strand) showing the regions covered by the 2′-O-Me oligos. (B) In vitro splicing of the U12-dependent P120 intron F in the presence of the indicated final concentrations of the antisense oligos. All reactions also contained an anti-U2 2′-O-Me oligo as described in Methods. The bands corresponding to the precursor RNA, the spliced exon product, the 5′ exon intermediate and the lariat intron product are indicated from top to bottom. Lanes 1,10,11 are from reactions containing only the anti-U2 oligo. (C) Splicing complexes formed in vitro on radiolabeled U12-dependent P120 intron F RNA in the presence of the indicated antisense 2′-O-Me oligos. All reactions also contained an anti-U2 2′-O-Me oligo. Complex H is a nonspecific complex, Complex A is the initial splicing-specific complex formed by addition of the U11/U12 snRNP, Complex B is formed by the addition of the U4atac/U6atac.U5 tri-snRNP to Complex A.
