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. 1997 Jun 10;94(12):6030–6035. doi: 10.1073/pnas.94.12.6030

Figure 1.

Figure 1

Splicing and crosslinking. (A) P120-4SU+2 with both the 5′ and 3′ phosphates of the 4SU+2 radioactive (lane 1) or uniformly labeled P120 (lane 2) substrate was used for splicing (30). The positions of the pre-mRNA, the spliced mRNA, the 2/3 intermediate, the free 5′ exon, and the lariat intron on an 8% polyacrylamide/8 M urea gel are indicated. ∗ indicates a degradation product that comigrates with the 2/3 intermediate (30). (B) A time course of crosslinking using site-specifically labeled P120-4SU+2 (lanes 1–10), P120-U+2 (lane 11), P120-4SU+4 (lanes 12–21), P120-U+4 (lane 22), P120-4SU+7 (lanes 23–32), or P120-U+7 (lane 33) substrate. A large splicing reaction containing each 4SU-substituted P120 was incubated and portions removed and irradiated with 365-nm UV light at the indicated times. Recovered RNAs were resolved on 5% polyacrylamide/8 M urea gels. Crosslinks 1, 2, and 3 are indicated by arrow heads. Lanes 8–11, 19–22, and 30–33 are 30-min controls where either UV irradiation (lanes 8, 19, 30), HeLa nuclear extract (lanes 9, 20, 31), or ATP (lanes 10, 21, 32) was omitted. (C) A parallel time course of splicing of uniformly labeled P120 pre-mRNA. At time points (lanes 1–7) identical to those chosen for irradiation (B), portions of the splicing reaction were removed. Recovered RNAs were resolved on an 8% polyacrylamide/8 M urea gel. The positions of the pre-mRNA, the spliced mRNA, the 2/3 intermediate, the free 5′ exon, and the lariat intron are indicated. A degradation product that comigrates with the 2/3 intermediate is denoted by ∗ (30).