Kinetics of splicing and splicing complex formation with Fushi tarazu (Ftz) and Zeste62 pre-mRNA. (A and D) Schematic representations of the Ftz-M3 and Zeste62-M3 pre-mRNA substrates. PY tract, polypyrimidine tract; nts, nucleotides. (B, C, E, and F) Splicing was performed with Ftz-M3 pre-mRNA (B and C) or Zeste62-M3 pre-mRNA (E and F) in Kc cell nuclear extract for the times indicated (in minutes) above each lane. In the experiments whose results are shown in panels B and E, 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. *, the band likely corresponds to debranched, spliced-out intron. Panels C and F show results for splicing complexes analyzed on a 3.5% native polyacrylamide gel and visualized by autoradiography. The positions of the H, A, B, and C complexes are indicated on the right.

EM of affinity-purified Drosophila B complexes reveals structural similarities with human B complexes. (A and B) EM overviews and single-particle images of negatively stained Drosophila B complexes formed on Ftz-M3 (A) or Zeste62-M3 (B) pre-mRNA in Kc nuclear extract. A schematic of the predominant view of Drosophila Ftz or Zeste62 B complex with the various regions/domains indicated is shown at the right. (C) Galleries of representative class averages of human B complexes formed on an adenovirus-derived pre-mRNA (top row), Drosophila B complexes formed on Ftz-M3 (middle row), or Zeste62-M3 (bottom row) pre-mRNA. For each class, approximately 40 to 50 individual images were averaged. The bar corresponds to 30 nm.

MS2 affinity selection of in vitro spliced and unspliced mRNPs. (A) Schematic representation of the Ftz-M3 pre-mRNA and FtzΔI-M3 mRNA. PY tract, polypyrimidine tract; nts, nucleotides. (B) FtzΔI-M3 or Ftz-M3 RNA was incubated for 180 min under splicing conditions in Kc cell nuclear extract, followed by oligonucleotide-directed cleavage by RNase H. Splicing substrates, intermediates, and products are indicated on the right. (C) Splicing reaction mixtures containing Ftz-M3 or FtzΔI-M3 were loaded onto a linear 10-to-30% (vol/vol) glycerol gradient containing G150 buffer. The radioactivity contained in each fraction was determined by Cherenkov counting. The profiles show the percentage of radioactivity in each fraction. Sedimentation coefficients (indicated by arrowheads) were determined by analyzing the UV absorbance of fractions of a reference gradient containing prokaryotic ribosomal subunits. (D) Gradient fractions 10 to 12 were pooled and subjected to MS2 affinity selection. Bound complexes were eluted with maltose, and RNA was recovered, separated by 12% denaturing PAGE, and visualized by silver staining (upper panel) or by autoradiography to detect radioactive species (lower panel). Lanes 1 and 4, 2.5% of the input (I) pooled gradient fractions. Lanes 2 and 5, 2.5% of the flowthrough (FT) of the amylose column. Lanes 3 and 6, 10% of the eluted (E) mRNPs. The positions of snRNAs, unspliced pre-mRNA, and splicing products are indicated on the right.

MS2 affinity selection of spliceosomal B complexes. (A) Splicing reaction mixtures containing Ftz-M3 pre-mRNA and Kc or HeLa nuclear extract or Zeste62-M3 pre-mRNA and Kc nuclear extract 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. (B) Gradient fractions 17 to 19 containing Ftz B complexes formed in Kc (lanes 1 to 3) or HeLa (lanes 4 to 6) extract or Zeste62-M3 B complexes formed in Kc extract (lanes 7 to 9) were 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). Lanes 1, 4, and 7, 2% of the input (I) pooled gradient fractions. Lanes 2, 5, and 8, 2% of the flowthrough (FT) of the amylose column. Lanes 3, 6, and 9, 25% of the eluted (E) spliceosomal complexes. The positions of the snRNAs, unspliced pre-mRNA, and splicing products/intermediates are indicated on the right.

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.