Initial recognition of U12-dependent introns requires both U11/5' splice-site and U12/branchpoint interactions

Abstract

We have investigated the formation of prespliceosomal complex A in HeLa nuclear extracts on a splicing substrate containing an AT-AC (U12-type) intron from the P120 gene. Using an RNase H protection assay and specific blocking oligonucleotides, we find that recognition of the 5' splice-site (5'ss) and branchpoint sequence (BPS) elements by U11 and U12 snRNPs, respectively, displays strong cooperativity, requiring both sites in the pre-mRNA substrate for efficient complex formation. Deletion analysis indicates that beside the 5'ss and BPS, no additional elements in the pre-mRNA are necessary for A-complex formation, although 5' exon sequences provide stimulation. Cross-linking studies with pre-mRNAs containing the 5'ss or BPS alone indicate that recognition of the BPS by the U12 snRNP is stimulated at least 20- to 30-fold by the binding of the U11 snRNP to the 5'ss in the same pre-mRNA molecule, whereas recognition of the 5'ss by U11 is stimulated approximately fivefold by the U12/BPS interaction. These results argue that intron recognition in the U12-dependent splicing pathway is carried out by a single U11/U12 di-snRNP complex, suggesting greater rigidity in the intron recognition process than in the major spliceosome.

Figure 1

Arrest of spliceosome assembly at the A complex stage. (A) Secondary structure model of RNA–RNA interactions between the pre-mRNA and U6atac/U12 snRNAs in the U12-dependent spliceosome (modified from Tarn and Steitz 1996b). The region of U6atac complementary to the U6atac_1–20 2′-O-methyl oligonucleotide is underlined. (B) Native gel analysis of splicing complexes formed on the U12-type P120 splicing substrate. (Left) Complex formation in a mock-treated splicing reaction; (right) complex formation in a reaction preincubated with 0.5 μm U6atac_1–20 2′-O-methyl oligonucleotide. (C) Northern analysis of the complexes detected in U12-dependent spliceosome assembly reactions. The reactions were preincubated either with or without the U6atac_1–20 2′-O-methyl oligonucleotide, as indicated. Following preincubation, unlabeled P120 splicing substrate was added, incubation was continued for 60 min, and the reactions were separated in a native gel and transferred to a membrane. U11 and U12 snRNAs were detected using full-length complementary riboprobes as indicated. (D) Secondary structure model of RNA–RNA interactions between the pre-mRNA and U6/U2 snRNAs in the U2-dependent spliceosome. The structure is based on the model of Madhani and Guthrie (1992), modified to include helix III suggested by Sun and Manley (1995). The region of U6 snRNA complementary to the U6_27–46 2′-O-methyl oligonucleotide is underlined. (E) Native gel analysis of splicing complexes formed on the U2-type adenovirus splicing substrate. (Left) Complex formation in a mock-treated splicing reaction; (right) complex formation in a reaction preincubated with 5 μm U6_27–46 2′-O-methyl oligonucleotide. The slowest migrating band present at later time points (80 and 100 min) has an intemediate mobility compared with complexes B and C (left).

Figure 2

Interactions of the P120 substrate analyzed by RNase H protection. (A) Schematic diagram showing the sites on the P120 splicing substrate targeted for RNase H clevage by five DNA oglionucleotides. (B) Protection of the P120 splicing substrate from RNase H hydrolysis after A complex assembly for 40 min. Either an individual site or two sites were targeted as indicated. The autoradiogram of a denaturing polyacrylamide gel shows an example of the RNase H hydrolysis pattern observed for the A complex. The graph (top) presents the mean values obtained in four independent experiments, which were normalized against the level of 5′ss protection, set at 100%. Standard deviation is indicated by error bars.

Figure 3

RNase H protection analyses in the presence of U11- or U12-specific blocking oligonucleotides. (A) Oligonucleotides designed to inhibit U11/5′ss and U12/BPS binding. (Top) Schematic diagram showing the base-pairing interaction between the U11 snRNA and the 5′ss of the P120 splicing substrate. The two PNA oligonucleotides used to inhibit the U11/5′ss interaction by forming a PNA₂–RNA triplex are indicated as lines above and below the U11 snRNA sequence. (Bottom) Schematic diagram showing the base-pairing interaction between the U12 snRNA and the BPS of the P120 splicing substrate. The U12_11–28 2′-O-methyl oligonucleotide used to inhibit the U12/BPS interaction is indicated as a line below the U12 snRNA sequence. The conserved nucleotides at the 5′ss and BPS are boxed (Dietrich et al. 1997). (B) Inhibition of P120 splicing by the U11-2P and U11-3P PNA oligonucleotides. Splicing reactions without substrate were preincubated in the presence of increasing concentrations of the two PNA oligonucleotides and the splicing activity was assayed 4 hr after addition of the P120 substrate by electrophoresis on a 5% denaturing polyacrylamide gel. Splicing intermediates and products are at right. (Asterisk) Degraded substrate fragment. (C) Effect of the U11-2P and U11-3P PNA oligonucleotides on U2-dependent splicing. Splicing reactions without substrate were preincubated in the presence of increasing concentrations of the two PNA oligonucleotides and splicing activity was assayed 1 hr after the addition of adenovirus substrate by electrophoresis on an 8% denaturing polyacrylamide gel. The lariat intermediate and product are indicated at right. (D) Effect of the 2′-O-methyl antisense oligonucleotide U12_11–28 (0.5 μm) on the RNase H protection profile of the P120 splicing substrate. After arrested A complex formation, individual sites were targeted as in Fig. 2A. (Bottom) Gel slices show recovery of the full-length P120 splicing substrate in the RNase H protection profile (see Fig. 2B). (Top) The graph shows mean values obtained from four independent experiments with the standard deviation indicated by error bars. (Solid bars) Protection observed on a mock-treated reaction; (open bars) reactions containg the U12_11–28 oligonucleotide; (shaded bars) control reactions in which splicing substrate was added at the time of the RNase H assay. (E) Effect of PNA oligonucleotides U11-2P and U11-3P (5 μm each) on the RNase H protection profile of the P120 splicing substrate. (Bottom) Gel slices show recovery of the full-length P120 splicing substrate in the RNase H protection profile (see Fig. 2B). (Top) The graph shows mean values obtained from three independent experiments, with the standard deviation indicated by error bars. The color coding is the same as in D.

Figure 4

Northern blot analysis of cross-linked products containing U12 or U11 and the full-length P120 splicing substrate or 3′ or 5′ half-substrate. Cross-linking conditions (±UV), RNA substrates, and antisense oligonucleotides used during preincubation are indicated (αU11—5 μm U11-2P and U11-3P; αU12—0.5 μm U12_11–28). The identities of the cross-linked bands are shown at right. (Asterisk) Cross-reacting bands detected in the absence of UV irradiation or splicing substrate. The sizes of DNA markers (M), a ³²P-labeled pBR322 digest, are at left. (A) Psoralen cross-linking of the unlabeled full-length P120 splicing substrate (lanes 1–5) or 3′ half-substrate (lanes 6–10). Cross-links were detected by hybridization with a U12-specific riboprobe. (Lanes 6–10) Exposure was eight times longer than that for lanes 1–5. (B) Cross-linking of a trace-labeled full-length P120 splicing substrate (lanes 1–6) or 5′ half-substrate (lanes 7–10) containing ^4SdU at position +7 of the intron. The full-length P120 control lacking the ^4SdU (lane 1) was not trace labeled. Cross-linked products were detected by hybridization with a U11-specific riboprobe. (C) Time course appearance of the lariat product in a standard splicing reaction containing either the full-length P120 splicing substrate (P120) or a substrate containing ^4SdU at intron position +7 (P120-^4SdU).

Figure 5

Northern blot analysis of cross-linked products containing U1 or U2 and the full-length adenovirus splicing substrate or 3′ or 5′ half-substrates. The cross-linking conditions (±UV), the RNA substrates, and antisense oligonucleotides used during preincubation are indicated (αU1—15 μm U1_1–14; αU2—4.8 μm U2_27–49; αU11—5 μm each U11-2P and U11-3P; αU12—0.5 μm U12_11–28). The identities of cross-linked bands are shown at right and the sizes of DNA markers, a ³²P-labeled pBR322 digest, at left. (A) Psoralen cross-linking of the unlabeled full-length adenovirus splicing substrate (lanes 1–7) or 5′ half-substrate (lanes 8–14). Cross-linked products were detected by hybridization with a U1-specific riboprobe. (B) Psoralen cross-linking of the unlabeled full-length adenovirus splicing substrate (lanes 1–7) or 3′ half-substrate (lanes 8–14). Cross-linked products were detected by hybridization with a U2-specific riboprobe. (Asterisk) Cross-reacting bands detected in the absence of UV irradiation or splicing substrate.

Figure 6

Psoralen cross-linking and arrested A complex formation on P120 deletion substrates. (A) Schematic diagram of the P120 deletion mutants. The sizes in nucleotides of the intron and exon portions are indicated. (B) Psoralen cross-linking of the unlabeled P120 splicing substrate and its deletion mutants after A complex assembly. Cross-linked products were detected by hybridization with a U12-specific riboprobe. The substrate used in each reaction is indicated at top. The identity of the cross-linked bands are indicated at right. (Asterisk) Cross-reacting bands detected in the absence of UV irradiation or splicing substrate. The multiple cross-linked species detected in lanes 4 and 5 are probably due to the multiple U12/BPS cross-links described by Tarn and Steitz (1996a). (C) Native gel analysis of A complex formation on the full-length P120 splicing substrate and its deletion mutants.