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. 2002 Nov 1;16(21):2778–2791. doi: 10.1101/gad.1030602

Figure 3.

Figure 3

Splicing and cross-linking of 4-thioU-containing RNAs. (A, upper panel) Splicing time courses of AdML-GG and AdML-AG RNAs containing single 4-thioU and 32P labels at position −24. Splicing precursors, intermediates, and products are indicated to the right. (Lower panel) Pattern of cross-linked proteins for each time point in upper panel. Molecular mass standards and apparent molecular weights of proteins cross-linking in a splicing-specific manner are indicated to the left and right, respectively. (B) Effects of nonspecific competitor RNA on cross-linking pattern for AdML-AG RNA. (Left panel) Following a 60-min incubation under splicing conditions, excess cold competitor RNA was added and reactions incubated for an additional 10 min before UV irradiation and RNase digestion. Open and closed arrows indicate bands that were sensitive or not, respectively, to addition of cold competitor RNA. (Right panel) Denaturing PAGE (15%) of splicing precursors and products in the presence (+) and absence (−) of cold competitor RNA showing that cross-linking differences in the left panel do not simply reflect a difference in the extent of splicing. (C) Glycerol gradient fractionation of splicing reactions containing site-specifically modified AdML-GG (lanes 1–7) or AdML-AG (lanes 9–15) substrates. Substrates were incubated for 90 min (AdML-GG) or 120 min (AdML-AG) and irradiated on ice prior to being loaded onto a 10%–30% glycerol gradient. Following glycerol gradient fractionation, fractions (numbers at top) were divided and either resolved by denaturing PAGE (upper panels) or RNase A digested and separated by SDS-PAGE (lower panels). Lane 8 corresponds to AdML control mRNA incubated for 45 min under splicing conditions, irradiated on ice, and directly resolved by denaturing PAGE (upper panels) or RNase A digested and separated by SDS-PAGE (lower panels), without prior glycerol gradient fractionation. Molecular mass markers and apparent molecular masses of splicing specific cross-linked proteins are indicated to the left and right of each gel, respectively. (D) Same as C except with site-specifically modified PIP.B control mRNA (lanes 1–7) and pre-mRNA (lanes 9–15).