Figure 2.

Sequences upstream of the branch site are essential for complex A assembly and formation of U2/U6 helix II within the trans-spliceosome. (A) Upstream mutations affect assembly of the Adeno 3′ RNA substrate into complex A. Nuclear extracts were incubated with ³²P-labeled Adeno 3′ RNA, or the UP-GG and UP-GA substrates (10 ng), with (+) or without (–) unlabeled Adeno 5′ SS oligonucleotide (440 ng), under trans-splicing conditions at 30°C for 20 min (G.Ast and A.M.Weiner, submitted for publication). RNP complexes were then resolved by native 4% PAGE. The positions of the H, A and B (U2/U4/U5/U6/3′ RNA) complexes are indicated on the left. Formation of complex A was both ATP- and temperature-dependent (data not shown). Left to right, lanes 1–6. (B) Upstream mutations affect the U2/U6 helix II interaction. After incubation as in (A), reactions were crosslinked with psoralen on ice, and the deproteinized RNAs resolved by denaturing 5% PAGE. The identities of crosslinked products obtained with the Adeno 3′ RNA substrate are indicated on the left (see Fig. 4) (70). The identity of the U2/BS crosslinks obtained with the UP-GG and UP-GA substrates was confirmed by RNase H protection with a DNA oligonucleotide complementary to U2 positions 28–42; cleavage of both complexes with a DNA oligonucleotide complementary to the 3′ SS served as a positive control (Fig. 4 and data not shown). The wild-type and mutant U2/BS crosslinks differ in size because the wild-type Adeno 3′ RNA substrate has 27 additional 5′ terminal nucleotides. Left to right, lanes 1–6.