Characterization of purified human Bact spliceosomal complexes reveals compositional and morphological changes during spliceosome activation and first step catalysis

Abstract

To better understand the compositional and structural dynamics of the human spliceosome during its activation, we set out to isolate spliceosomal complexes formed after precatalytic B but prior to catalytically active C complexes. By shortening the polypyrimidine tract of the PM5 pre-mRNA, which lacks a 3' splice site and 3' exon, we stalled spliceosome assembly at the activation stage. We subsequently affinity purified human B(act) complexes under the same conditions previously used to isolate B and C complexes, and analyzed their protein composition by mass spectrometry. A comparison of the protein composition of these complexes allowed a fine dissection of compositional changes during the B to B(act) and B(act) to C transitions, and comparisons with the Saccharomyces cerevisiae B(act) complex revealed that the compositional dynamics of the spliceosome during activation are largely conserved between lower and higher eukaryotes. Human SF3b155 and CDC5L were shown to be phosphorylated specifically during the B to B(act) and B(act) to C transition, respectively, suggesting these modifications function at these stages of splicing. The two-dimensional structure of the human B(act) complex was determined by electron microscopy, and a comparison with the B complex revealed that the morphology of the human spliceosome changes significantly during its activation. The overall architecture of the human and S. cerevisiae B(act) complex is similar, suggesting that many of the higher order interactions among spliceosomal components, as well as their dynamics, are also largely conserved.

FIGURE 1.

Truncation of the polypyrimidine tract (PPT) stalls splicing prior to the first catalytic step. (A) Schematic of PM5 pre-mRNA constructs used, where the polypyrimidine tract was truncated to the indicated length. All constructs have a 6-nt stretch between the branch point adenosine and PPT. Kinetics of in vitro splicing (B,D), and splicing complex formation (C,E) with the PM5 pre-mRNA and truncated versions thereof. Pre-mRNAs were incubated under splicing conditions in the presence of HeLa nuclear extract for the times indicated above each lane. RNA was analyzed by denaturing PAGE and visualized by autoradiography (B,D) The pre-mRNA and splicing intermediates are indicated on the right or left. Spliceosomal complex formation was assayed on a native agarose gel. The positions of the H/E, A, B, C, and B^act complexes are indicated. The arrow indicates the position of the B^act complex. Asterisk, unknown contaminating band.

FIGURE 2.

Affinity purification of human, spliceosomal B^act complexes. (A) Glycerol gradient fractionation of splicing complexes formed on the PM5-20 construct. In vitro splicing, followed by RNAse H digestion, was performed as described in the Materials and Methods. Complexes were then separated on a linear 10%–30% glycerol gradient and the ³²P-RNA in each fraction was determined by Cherenkov counting. S-values were determined by comparison with a reference gradient containing prokaryotic ribosomal subunits. Gradient profiles for both PM5-10 (not shown) and PM5-20 complexes were identical. (B,C) Composition of affinity-purified complexes. Gradient fractions containing 45S complexes (fractions 17–21) were pooled and subjected to MS2 affinity selection. (B) RNA was recovered, separated by denaturing PAGE, and visualized by autoradiography (lanes 1 and 2) and by silver staining (lanes 3 and 4). The positions of the snRNAs and the PM5-20 and PM5-10 pre-mRNA are indicated on the right. Asterisk, RNase H degradation product. The other minor bands seen in lanes 1 and 2 do not correspond to splicing intermediates, but are likely degradation products. (C) Protein was recovered from the affinity purified B^act complexes (lanes 1 and 2), analyzed by SDS-PAGE on an 8%/14% polyacrylamide step gel, and visualized by staining with Coomasie. The size of molecular weight markers (lane 3) is indicated on the right.

FIGURE 3.

Immunoblotting reveals the dynamics of spliceosomal protein association and post-translational modification of SF3b155 and CDC5L. (A) B and C complexes formed on PM5 pre-mRNA, and B^act complexes formed on PM5-20 pre-mRNA, were affinity purified and their proteins analyzed by Western blotting with the antibodies indicated below or in (B) against SF3b155 or CDC5L. Antibodies against MS2-MBP and the U5-116k protein were used to ensure equal loading. (C) B^act and C complexes were incubated in the absence (−) or presence (+) of calf intestine pyrophosphatase (CIP) directly (left two lanes) or after first incubating at 96°C to inactivate endogenous enzymatic activity (right two lanes). Immunoblotting was performed with anti-SF3b155 or anti-CDC5L antibodies, as indicated below.

FIGURE 4.

Negative stain electron microscopy of affinity purified B^act complexes. (A) Negative stain EM raw images of human B^act complexes formed on the PM5-20 pre-mRNA substrate. (B) Selected class averages of spliceosomal B^act complexes assembled on the PM5-20 pre-mRNA construct. At the right, a schematic drawing corresponding to class averages seen in columns 1–4 is shown. Bar = 50 nm.

FIGURE 5.

Comparison of the EM images of the human B and B^act complexes. Typical class averages of the human B (left panels) (Deckert et al. 2006) and B^act (right panels) complexes are shown. Bar = 50 nm.

FIGURE 6.

The human and yeast B^act complexes share a similar morphology. Typical class averages of the human (right panels) and yeast (left panels) B^act complexes (Fabrizio et al. 2009), visualized by electron microscopy after negative staining, are shown. Bar = 50 nm.