Base pairing interactions between U4/U4atac and U6/U6atac snRNAs. (A) Base pairing model of the human U4/U6 di-snRNP showing the sequences and locations of the stem I and stem II helices. (B) Base pairing model of the human U4atac/U6atac di-snRNP showing the sequences and locations of the stem I and stem II helices (29). (C) Base pairing model showing the proposed interactions of human U4 snRNA with a mutant human U6atac snRNA containing the U6 ISL sequence (7). The mutations introduced into the U6atac snRNA are shown in bold. (D) Base pairing model showing the proposed interactions of the U6atac Ath ISL mutant and the compensatory mutant U4 Ath Supp snRNA described in the text. The mutations introduced into U6atac and U4 snRNAs are shown in bold type. Also shown are the location and sequences of the U4 Stem I mutants described in the text.

Psoralen crosslinking of snRNAs in vitro. Labeled U6atac Ath ISL RNA was incubated with unlabeled U4, U4 Ath Supp, or U4atac snRNA as indicated and treated with UV light in the presence or absence of psoralen. Samples were analyzed by denaturing polyacrylamide gel electrophoresis. The positions of labeled U6atac Ath ISL RNA and a slower migrating crosslinked species are indicated.

Modulation of spliceosome choice at a bifunctional 5′ splice site. (A) Splicing pattern of the P120 A1G/C99G mutant. The two splice site mutations are shown in bold. The 5′ GU splice site is joined to either a U12-dependent 3′ splice site AG (–1) or a U2-dependent 3′ splice site AG (–6). (B) RT-PCR analysis of the in vivo splicing of the P120 intron F in transfected Chinese hamster ovary cells. Lane 1 shows the splicing of wild-type P120; lane 2 shows the splicing pattern of the P120 A1G/C99G mutant. The lower band represents U12-dependent splicing to the wild-type 3′ splice site, whereas the upper band represents U2-dependent splicing to the cryptic 3′ splice site at –6. Lanes 3–6 show the effect of adding 2, 5, 10, and 15 μg of a wild-type U4 snRNA expression construct to the transfections. Lanes 7–10 show the effect of adding the same levels of a U4 snRNA expression construct containing the U4atac stem II interaction sequence. Lanes 11–14 show the effect of adding the same levels of a U4 snRNA expression construct containing the Ath stem II interaction sequence (U4 Ath Supp). The use of the U2- and U12-dependent 3′ splice sites is shown below each lane as a percentage of the total spliced signals. Also shown is the ratio of U2- to U12-dependent splicing in each lane.

In vivo suppression assay of mutant snRNAs. RNA was extracted from cells transiently transfected with the P120 minigene and snRNA constructs as shown. The splicing pattern of the U12-dependent P120 intron F was analyzed by RT-PCR amplification using primers in the adjacent exons. The positions of bands corresponding to unspliced and correctly spliced products are shown as well as the cryptic spliced product. The cryptic spliced band is caused by splicing between a pair of U2-dependent splice sites located within the P120 F intron. The constructs used for the various lanes are: lane 1, mock transfected cells; lane 2, empty pCB6 expression vector; and lane 3, wild-type P120 minigene. Lanes 4–13 used the P120 5′ splice site mutant CC5/6GG and the following snRNA expression constructs: lane 4, the P120 mutant alone; lane 5, U11 GG6/7CC; lane 6, U6atac GG14/15CC Ath ISL; lane 7, U6atac GG14/15CC Ath ISL plus U11 GG6/7CC; lane 8, U6atac GG14/15CC Ath ISL plus U4atac Ath Supp plus U11 GG6/7CC; lane 9, U6atac GG14/15CC Hu U6 ISL plus U11 GG6/7CC; lane 10, U6atac GG14/15CC Hu U6 ISL plus U4atac Hu U6 Supp plus U11 GG6/7CC; lane 11, U6atac GG14/15CC Ath ISL plus U4 Ath Supp plus U11 GG6/7CC; lane 12, U6atac GG14/15CC Ath ISL plus U4 Ath Supp SI-1 plus U11 GG6/7CC; and lane 13, U6atac GG14/15CC Ath ISL plus U4 Ath Supp SI-2 plus U11 GG6/7CC. Lane M contains molecular size markers.