Fig 1.
Flowchart of the procedure designed for detecting the release of 35S U5 snRNPs from the B* complexes during in vitro splicing.
Splicing complexes assembled on the radioactively labelled pre-mRNA were precipitated with the matrix-coupled anti-SKIP antibodies, and eluted by competition with the cognate peptide. Eluted complexes were subjected to the second immunoprecipitation with the beads coupled with anti-SF3a66K antibodies. The complexes bound to the beads were incubated under splicing conditions in the presence of MN-treated HeLa nuclear extract. The complexes released into the supernatant were analysed by gradient density centrifugation.
Fig 2.
The activated spliceosomes immobilised to the beads are capable of catalysing both trans-esterification reactions in the presence of MN-treated nuclear extract.
(A) The Cherenkov counting analysis of the material released from the beads under splicing condition (1—in the presence of ATP at 30°C) and under conditions, which do not support splicing (2—in the absence of ATP at 30°C; 3—in the presence of ATP at 0°C). (B) The RNA extracted from both the supernatants (lane 2, 3, 4) and the beads (lane 1, 5, 6, 7) after incubation under splicing conditions (lane 2 and 5) and under conditions which do not support splicing (lane 3, 4 and 6, 7), was analysed by denaturing PAGE followed by autoradiography. Lane 1 contains the RNA bound to the beads prior to the incubation in the presence of MN-treated nuclear extract. Identities of the RNA species are shown on the right.
Fig 3.
Characterisation of the complexes released into the supernatant from immobilised activated spliceosomes during splicing in the presence or absence of ATP by glycerol gradient centrifugation.
(A) The RNA extracted from each gradient fraction was fractionated by denaturing PAGE and the 32P-containing species were detected by autoradiography. S-values were determined by comparison with the reference gradients containing 30S and 50S ribosomal subunits. The RNA identities are shown on the right. (B) Northern blot analysis of gradient fractions from panel A with the U5 snRNA specific probe. Fractions corresponding to the 35S U5 snRNP and 45S B* spliceosomes are underlined. (C) Quantification of the signals corresponding to the U5 snRNA (panel B) using a PhosphorImager. Open circles correspond to the reaction carried out in the presence of ATP and closed circles—in the absence of ATP.
Fig 4.
Immunoprecipitation of the gradient purified 45S and 35S complexes using anti-DDX35 antibodies.
The fractions from the gradient analysed in Fig 3, corresponding to the 35S region (fractions 9–12—lane 3 and 4) or the 45S region (fractions 14–17—lane 1 and 2), were combined and subjected to immunoprecipitation with the anti-DDX35 antibodies (lane 1 and 3) or with the anti-DDX35 antibodies pre-blocked with antigenic peptide (lanes 2, 4). The RNA, extracted from the beads, was labelled with 32P-pCp and analysed by denaturing PAGE followed by autoradiography. Identities of the RNA species are shown on the left. The bands marked 5.8S and 5S correspond to rRNA that was precipitated unspecifically.