(A) Eight proteins cross-link to 4-thioU-substituted WT-72. 4-thioU-substituted ³²P RNAs were transcribed in vitro as described in Materials and Methods. The 5′ ss mutant (5′ ss mut) contains a mutated 5′ splice site (AUGUAU; mutated nucleotides are underlined). CCs were formed either with an HA-tagged extract (U1–70K–HA) or a nontagged wild-type extract (Y59, WT, lane 4). After incubation at 25°C for 30 min, complexes were irradiated with 365 nm UV light at 4°C for 5 min. For lanes 1–5, immunoprecipitation was then done with either anti-HA (12CA5) or anti-Nam8p antibodies as indicated. After immunoprecipitation, beads were treated with RNase T1/A for 20 min at 37°C. Beads were boiled in SDS-polyacrylamide sample buffer, and samples were analyzed on a Tris-HCl 4%–20% linear gradient polyacrylamide gel and autoradiographed. The samples in lanes 6 and 7 were from the same experiment as lanes 1 and 2 but without immunoprecipitation. The extract was treated with RNase T1/A before SDS-PAGE. (B) Comparison of the cross-linking pattern of WT-72 with that of WT-B. The substrate in lane 1 is WT-72, as in A. The substrate in lane 2 is a ligation product of a 5′ RNA and a 3′ RNA. The 5′ RNA is 4-thioU-substituted ³²P-labeled WT-72. The 3′ RNA is a nonradioactive non-4-thioU-substituted RNA. It was transcribed in vitro using a PCR product as template that carried a T7 promoter and sequences downstream of WT-72 as shown in Fig. 1A. The ligation product was gel purified and used for cross-linking as in A. (C) Cross-linking patterns of WT-72 substrate irradiated with either 254 nm (lanes 2,3) or 365 nm UV light (lanes 1,4). RNA substrates in lanes 3 and 4 were radiolabeled but not 4-thioU substituted.

Exon and intron mapping. (A) Schematic representation of the three cross-linking substrates. 5′ ss sequences are shown in a large font. (B) Identification of proteins that bind within or downstream of the 5′ ss region (lane 2) or within or upstream of the 5′ ss region (lane 3). Experiments were done as in Fig. 2A. All splicing extracts were wild type, and the antibody was anti-Nam8p. Proteins are indicated at left.

High-resolution intron mapping. (A) Schematic representation of RNA sequences. Intron Us are numbered at top of WT-72 and underlined; 4-thioUs are in boldface type. The 5′ ss sequences are in large font. (B) Cross-linking profiles. Experiments were done as in Fig. 2A. The splicing extract was wild type, and the antibody was anti-Nam8p. Nam8p and Snu56p, which are very faint in lane 2 and less prominent in lane 4, are indicated at left.

Model of the pre-mRNA–protein interactions in the CC1 complex. For U1 snRNA, only the 5′ arm, stem/loop I, II, and III are shown. The 5′ ss region sequences are shown in lowercase and boldface letters. The 5′ arm sequence of U1 snRNA is shown in uppercase letters. The intron sequences downstream of 5′ ss are abbreviated by a thin line; exon sequences are abbreviated by a rectangle and labeled. U1 snRNP-specific proteins are shown in red, Sm proteins in blue, and CBC proteins in green.

WT-72 is functional for CC formation. (A) Full-length WT-B sequence. WT-B pre-mRNA was transcribed in vitro from the DdeI-linearized plasmid pRS195 and is 195 nucleotides in length. WT-72 was transcribed in vitro from the SalI-linearized plasmid and is 72 nucleotides in length. These two restriction sites are indicated (↓). Positions of 4-thioU in WT-72 are in boldface type. The 5′ss, branchpoint, and 3′ splice site regions are in larger font and underlined. \ indicates the splice sites. The 5′ exon sequence has negative numbers, and the intron sequence has positive numbers. (B) Competition assay. Radiolabeled WT-B was mixed with increasing amounts of cold competitor RNA, WT-B (top), or WT-72 (bottom) before the addition of a U2-depleted wild-type extract. After incubation at 25°C for 20 min, the complexes were analyzed by native gel electrophoresis. Nonspecific competitor RNA was without effect. (C) Quantification of competition experiment in B. Bands corresponding to the CC region of the gel shown in B were quantified and plotted against competitor RNA concentration (♦) WT-B; (█) WT-72. Background value was defined as 0% CC, and the CC formed without competitor was defined as 100% CC. (D) Native gel analysis of WT-72. Radioactively labeled WT-B (lane 2) and WT-72 (lane 1) were incubated in a U2-depleted wild-type Y59 extract at 25°C for 20 min. CCs were analyzed by native gel electrophoresis. Arrows indicate the position of CC1 and CC2.

Identification of the eight cross-linked proteins. Complex formation, cross-linking, and all analyses were as in Fig. 2A. The splicing extract used in each experiment is indicated at the top of the lane. All cross-linking was performed with radiolabeled and 4-thioU-substituted WT-72, and immunoprecipitations were with the anti-Nam8p antibody (except for lanes 4,5,6 in D, in which anti-U1–70K antibody was used). All samples were run on Tris-HCl 4%–20% linear gradient polyacrylamide gels, except for the samples in lanes 6 and 7 in A and lanes 1, 2, and 3 in D. In these cases, the gel concentrations were 7.5% and 10%–20%, respectively. In the case of A, lanes 6 and 7, proteins 2–8 were run off the gel. The bands corresponding to the identified proteins are marked by arrows or arrowheads. (A) yCBP80; (B) U1–C; (C) U1–70K; (D) SmD1 and SmD3; (E) Nam8p and Snu56p. The * in D indicates that the SmB assignment is tentative, as described in the text.

High-resolution exon mapping. (A) Schematic representation of RNA sequences. The 5′ exon Us are numbered at top of sequences. The 5′ ss sequences are shown in a large font. (B) Cross-linking profiles. Experiments were as in Fig. 2A. Splicing extracts were wild type, and the antibody was anti-Nam8p. Proteins are indicated at left.

Cross-linking to the 5′ ss region uridines. (A) Schematic representation of the three site-specifically labeled 4-thioU substrates. (*) The position of the single ³²P. Boldface type indicates the adjacent position of the single 4-thioU in each substrate. The 5′ phosphate of the 4-thioU is labeled. (B) Experiments were done as in Fig. 2A. The three RNAs, +2U, +4U, and +6U are ligation products of a 5′ RNA and a 3′ RNA. The 5′ RNAs was a capped, nonradioactive RNA from in vitro transcription. The 3′ RNAs were chemically synthesized with a single 4-thioU at their 5′ ends. The 5′ end of the 3′ RNA was kinased with [α-³²P]ATP and ligated to the 5′ RNA (Moore and Query 1998). Corresponding extracts and RNAs are indicated at top of each lane . The position of U1–C is marked at right.