Fig. 1. Sequence alignment of the human 65 kDa protein with homologous proteins. The human 65 kDa protein is shown aligned by the Clustal method with the corresponding proteins from C.elegans, S.pombe and S.cerevisiae (Sad1p). Residues identical to those in the 65 kDa sequence are shown on a black background, and conserved residues (grouped DE, KRH, AFILMPVW and CGNQSTY) are shaded yellow. RS, RD and RE dipeptides in the N-terminal part of the sequence are shown on a green field. The domains are indicated above the sequences, as follows: ZnF, ubiquitin C-terminal hydrolase-like zinc finger; UCH-1 and UCH-2, ubiquitin C-terminal hydrolases family 2. Consensus residues that attribute the sequence to a particular domain are shown below each domain.

Fig. 2. Sequence alignment of the human 110 kDa protein with homologous proteins. The human 110 kDa protein is shown aligned by the Clustal method with the corresponding proteins from D.melanogaster, C.elegans, N.crassa, P.falciparum, S.pombe and S.cerevisae (Snu66p). Residues identical in at least four sequences are shown on a black background, and conserved residues, grouped as in Figure 1, are shaded yellow. RS, RD and RE dipeptides in the N-terminal part of the sequence are in green.

Fig. 3. Authenticity of the cDNAs encoding the 65 and 110 kDa proteins. Lane 1: proteins of purified U4/U6⋅U5 tri-snRNPs (indicated on the left) were separated by 10% SDS–PAGE and stained with Coomassie blue. Lanes 2 and 3: the 65 kDa (lane 2) or 110 kDa (lane 3) proteins, prepared by translation in vitro from the corresponding cDNAs in the presence of [³⁵S]methionine, were analysed by 10% SDS–PAGE and made visible by fluorography. Lanes 4–11: western blot of HeLa nuclear extracts (lanes 4, 6, 8 and 10; NE) and purified U4/U6⋅U5 tri-snRNPs (lanes 5, 7, 9 and 11; tri-snRNP) probed with antisera against the 65 kDa (lanes 6 and 7; α-65) or 110 kDa protein (anti-C110) (lanes 10 and 11; α-110). Control experiments with the corresponding pre-immune sera are shown in lanes 4 and 5, and 8 and 9, respectively (NIS). Molecular weight markers are shown on the right.

Fig. 4. Immunoprecipitation of snRNPs associated with the 65 or the 110 kDa protein. SnRNP particles were precipitated from HeLa nuclear extract, at the salt concentrations indicated above the lanes, by using anti-116 kDa (lanes 1–3), anti-pep 110 kDa (lanes 7–9), anti-65 kDa (lanes 13–15) and the pre-immune serum corresponding to anti-pep 110 kDa (lanes 4–6) or anti-65 kDa (lanes 10–12). Co-precipitated RNAs were extracted, labelled with [³²P]pCp, fractionated by 10% urea–PAGE and detected by autoradiography. The positions of snRNAs are indicated on the left. Since U6 snRNA is not labelled efficiently with [³²P]pCp (Lund and Dahlberg, 1992), its presence in the various immunoprecipitates was detected by northern blot hybridization (lower panel).

Fig. 5. The antibodies against the 65 kDa (A) or the 110 kDa protein (B) inhibit splicing in vitro. Splicing reactions containing different amounts of the protein A-purified anti-65 kDa or anti-110 kDa antibody (anti-C110) (lanes 7–11), or corresponding IgGs from pre-immune serum (lanes 2–6), were incubated for 60 min and analysed on a 14% denaturing polyacrylamide gel. The amount of added antibody is stated above each lane. Lane 1 shows the pre-mRNA added to each splicing reaction. Pre-mRNA, splicing intermediates and products are indicated schematically.

Fig. 6. Immunodepletion of the 65 or the 110 kDa protein does not disrupt the U4/U6⋅U5 tri-snRNPs. (A) Western blot of mock-depleted extracts (lanes 2 and 6), 65 kDa-depleted extracts (lanes 3 and 8) and 110 kDa-depleted extracts (lanes 4 and 7) probed with anti-65 kDa or anti-110 kDa antibodies as indicated above each lane. In lanes 1 and 5, total snRNP proteins were blotted as a control. The bands correspond ing to the 65 and 110 kDa proteins are indicated. The bands corres ponding to the uncharacterized protein recognized by the anti-65 kDa antibody are marked with asterisks. (B) Immunoprecipitation of snRNPs with anti-U5 116 kDa antibodies from the mock-depleted (lane 1), 65 kDa-depleted (lane 2) and 110 kDa-depleted (lane 3) extracts. RNAs were isolated and identified by northern blot analysis.

Fig. 7. The 65 kDa protein is required for splicing in vitro. (A) Immunodepletion of the 65 kDa protein from HeLa nuclear extract inhibits splicing in vitro. The time course of splicing reactions was monitored in 65 kDa-depleted (lanes 5–8) and mock-depleted extracts (lanes 1–4). The bands corresponding to pre-mRNA, intermediates and spliced products are indicated on the left. (B) Splicing activity of the depleted extract is restored by addition of the recombinant 65 kDa protein produced in E.coli. Splicing reactions containing the mock-depleted extract (lane 1), or 65 kDa-depleted extracts complemented with 0, 34 and 68 ng of the recombinant protein (lanes 2, 3, and 4), were incubated for 60 min and analysed as described in Materials and methods. (C) Recombinant 65 kDa protein promotes the transition from the A to B complex in the depleted extracts. The spliceosome assembly was analysed by native gel electrophoresis in mock-depleted extracts (lanes 1–4), 65 kDa-depleted extracts (lanes 5–8) and 65 kDa-depleted extracts complemented with 34 ng of recombinant 65 kDa protein (lanes 9–12). The bands corresponding to the H, A and B complexes as well as the gel origin are indicated on the left.

Fig. 8. The 110 kDa protein is required for splicing in vitro. (A) Immunodepletion of the 110 kDa protein from HeLa nuclear extract inhibits splicing in vitro. The time course of splicing reactions was monitored in 110 kDa-depleted (lanes 5–8) and mock-depleted extracts (lanes 1–4). The bands corresponding to pre-mRNA, inter mediates and spliced products are indicated on the left. (B) Splicing activity of the depleted extract is restored by addition of the recombin ant 110 kDa protein expressed in E.coli. Splicing reactions containing the mock-depleted extract (lane 1), or 110 kDa-depleted extracts complemented with 0, 25, 50 and 100 ng of the recombinant protein (lane 2, 3, 4 and 5), were incubated for 60 min and analysed as described in Materials and methods. (C) The recombinant 110 kDa protein promotes the transition from the A to B complex in the depleted extracts. The spliceosome assembly was analysed by native gel electrophoresis in the mock-depleted extract (lanes 1–4), the 110-kDa-depleted extract (lanes 5–8) and the 110 kDa-depleted extract complemented with 50 ng of the recombinant 110 kDa protein (lanes 9–12). The bands corresponding to the H, A and B complexes as well as the gel origin are indicated on the left.