FIGURE 2.

(A) Schematic diagrams of the smE-CFP (splicing reporter) and the U4atac overexpression constructs. (Top) The smE-CFP construct is controlled by the CMV promoter and contains a portion of mouse smE (exons 1–3) fused to the cyan fluorescent protein (CFP) portion of the reporter construct described by Patel et al. (2002). The lengths of the exons and introns and the intron type (U12- or U2-type) are indicated. (Bottom) Schematic diagram of the U4atac overexpression construct. The coding sequence of the U4atac snRNA gene and 3′ box downstream of the coding region were fused with the U12 snRNA promoter. In both constructs the transcription start site is indicated by an arrow. (B) Effect of U4atac overexpression on splicing in 3T3-D1 and L-929 cells. Total RNA (8.5 μg) from transiently transfected 3T3-D1 (lanes 1–3) and L-929 (lanes 8–10) cells was analyzed by Northern blotting using a probe specific to smE-CFP exons. DNase treated lanes (4, 5 and 11, 12) contained approximately 4 μg of total RNA. Cells were transfected with smE-CFP alone, with U4atac alone, or with both constructs as indicated above each lane. In lanes 4–7 and 11–14, the samples were treated with either DNase or RNase as indicated, to confirm that the observed signal was derived from RNA. The migration of the reporter transcript with either unspliced or spliced U12-type intron 1 is indicated on the left. (Bottom) Ethidium bromide-stained gel from the same experiment showing 28S and 18S rRNAs as a loading control. (C) RT-PCR analysis of the effect of U4atac snRNA overexpression in 3T3-D1 and L-929 cells. The migration of the PCR products with either unspliced or spliced U12-type intron 1 is indicated on the left.