FIG. 4.

Affinity purification of Drosophila C complexes. (A) Schematic representation of the M3-Ftz-longPYT pre-mRNA substrate. PY tract, polypyrimidine tract; nts, nucleotides. (B) Splicing was performed with M3-Ftz-longPYT pre-mRNA in Kc cell nuclear extract for the times indicated (in minutes) above each lane. In lanes 8 and 9, DNA oligonucleotides complementary to the Ftz 5′ exon (nt +6 to +17 and nt +18 to +30 relative to the 5′ ss) were added after 10 min or 180 min of splicing, and the reaction mixture was incubated for an additional 20 min to allow RNase H digestion of early spliceosomal complexes. RNA was recovered, analyzed on a 12% polyacrylamide-8 M urea gel, and visualized by autoradiography. Splicing substrates, intermediates, and products are indicated on the right. *, RNase H digestion product. (C) Splicing complexes were analyzed on a 3.5% native polyacrylamide gel and visualized by autoradiography. The positions of the H, A, B, and C complexes and RNase H digestion products (*) are indicated on the right. (D) RNA composition of MS2-affinity-purified C complexes. Gradient containing Ftz C complexes formed in Kc extract (fractions 19 to 22) was pooled and subjected to MS2 affinity selection. Bound complexes were eluted with maltose, and RNA was recovered, separated by denaturing PAGE, and visualized by silver staining (upper panel) or by autoradiography (lower panel). The positions of the snRNAs, unspliced pre-mRNA, and splicing products/intermediates are indicated on the right. (E) Splicing reaction mixtures containing Ftz-M3 pre-mRNA or M3-Ftz-longPYT pre-mRNA were subjected to glycerol gradient centrifugation. The radioactivity contained in each gradient fraction was determined by Cherenkov counting and expressed as the percentage of the total ³²P-RNA loaded onto the gradient. Sedimentation coefficients, indicated by arrowheads, were determined by analyzing the UV absorbance of fractions of a reference gradient containing prokaryotic ribosomal subunits.