Fig. 2

Transcripts Containing Minor Introns Leave the Nucleus, and the Minor Spliceosome Acts in the Cytoplasm

(A) Distribution of major- and minor-class intron-containing transcripts after nucleo-cytoplasmic fractionation of NIH 3T3 cells. Nuclear (n) and cytoplasmic (c) fractions were obtained as described in the legend to Figure 1D. Splicing of U2-dependent intron G and the U12-dependent intron F in endogenous P120 pre-mRNA was monitored by RT-PCR using primers as indicated in the scheme (open/gray boxes, exons; lines, introns; arrows, primers). The number of amplification cycles performed is indicated on top. In lanes 9 and 10, 36 cycles were performed. Lanes 7 and 8 correspond to RT-PCR reactions lacking reverse transcriptase. Splicing products are diagrammed on the right (exons, open boxes; introns, lines). M, 100 base pair (bp) DNA ladder; lowest band is 100 bp.

(B) Nucleo-cytoplasmic distribution of transcripts from several genes with minor introns upon transfection of NIH 3T3 cells with either a control (cMO) or an antisense-U6atac morpholino oligomer (U6atacMO). RT-PCR analysis of splicing of the single minor-class intron (U12) and of one of the major-class introns (U2) are shown. P120, nucleolar proliferation-associated antigen P120 (U12, intron 6-7; U2, intron 7-8); adprt, poly-ADP-ribosyl transferase (U12, intron 22-23; U2, intron 21-22); and e2f1, E2F1 transcription factor (U12, intron 4-5; U2, intron 2-3). Primers hybridized in the upstream and downstream exons, respectively. For e2f1, an additional intronic forward primer was used. Symbols are as in (A).

(C) Confocal fluorescence microscopy of living NIH 3T3 cells transfected with a fluorescein-modified antisense-U6atac morpholino (0.25 nmol) conjugated to either a wild-type NES peptide (NES-U6atac-FL) or a mutated, inactive NES peptide (mNES-U6atac-FL). DNA counterstaining was performed by DRAQ5.

(D) Real-time quantitative RT-PCR analysis of intron F splicing from endogenous P120 pre-mRNA. NIH 3T3 cells were mock transfected or transfected with a U6atac morpholino oligomer (0.25 nmol) conjugated to either the wild-type NES peptide (NES-U6atac) or the mutated NES peptide (mNES-U6atac). RNA was prepared 4 hr after transfection, and relative expression levels of spliced and unspliced RNA were determined. Values in the graph are expressed as relative percentage of unspliced RNA (set to 100% in transfections without morpholino conjugate). Error bars indicate standard deviations from three independent transfections.