Rearrangements within human spliceosomes captured after exon ligation

Abstract

In spliceosomes, dynamic RNA/RNA and RNA/protein interactions position the pre-mRNA substrate for the two chemical steps of splicing. Not all of these interactions have been characterized, in part because it has not been possible to arrest the complex at clearly defined states relative to chemistry. Previously, it was shown in yeast that the DEAD/H-box protein Prp22 requires an extended 3' exon to promote mRNA release from the spliceosome following second-step chemistry. In line with that observation, we find that shortening the 3' exon blocks cleaved lariat intron and mRNA release in human splicing extracts, which allowed us to stall human spliceosomes in a new post-catalytic complex (P complex). In comparison to C complex, which is blocked at a point following first-step chemistry, we detect specific differences in RNA substrate interactions near the splice sites. These differences include extended protection across the exon junction and changes in protein crosslinks to specific sites in the 5' and 3' exons. Using selective reaction monitoring (SRM) mass spectrometry, we quantitatively compared P and C complex proteins and observed enrichment of SF3b components and loss of the putative RNA-dependent ATPase DHX35. Electron microscopy revealed similar structural features for both complexes. Notably, additional density is present when complexes are chemically fixed, which reconciles our results with previously reported C complex structures. Our ability to compare human spliceosomes before and after second-step chemistry has opened a new window to rearrangements near the active site of spliceosomes, which may play roles in exon ligation and mRNA release.

FIGURE 1.

A short 3′ exon pre-mRNA substrate stalls human spliceosomes between the second step of splicing and mRNA release. (A) Denaturing PAGE analysis of in vitro splicing reactions with radiolabeled pre-mRNA substrates containing a mutated 3′ splice site and a 51-nt 3′ exon (mut 3′ ss), wild-type 3′ splice site and a 51-nt 3′ exon (wt 3′ ss), or wild type 3′ splice site and a 19-nt 3′ exon (short 3′ exon). Reaction time points, in minutes, are noted at the top of the gel, and splicing intermediates are indicated on the left as, from top to bottom, lariat-intron intermediate, lariat intron, pre-mRNA, mRNA, and 5′ exon. The different positions of pre-mRNA, lariat-intron intermediate, and mRNA with 51- vs. 19-nt 3′ exons are indicated by brackets. (B) Glycerol gradient (10%–30%) sedimentation of splicing reactions using the same pre-mRNA substrates as in A. All three panels show denaturing PAGE analysis of fractions collected from top to bottom of the gradient (1–16, respectively). Quantification of indicated band intensities vs. fraction number is graphed at the right. (C) Native gel analysis of splicing reactions correlating to those shown in A. The position of H/E, A, B, C/C-like, and mRNP complexes are indicated on the left.

FIGURE 2.

RNase H protection mapping of mRNA in the post-catalytic spliceosome complex. (A) Sequence of mRNA with short 3′ exon (gray shaded) and DNA oligos used for RNase H digestion shown in B. (B) Denaturing PAGE analysis of splicing reactions using a pre-mRNA substrate with a short 3′ exon to which DNA oligos were added after 60 min to initiate RNase H digestion of complementary RNA sequences. Oligos targeted the mRNA diagrammed at the top in the 5′ exon (lanes 1–9), exon junction of the mRNA (lane 10), and 3′ exon (lane 11). Lane U is a splicing reaction in the absence of added DNA oligo. Splicing intermediates are indicated on the left as, from top to bottom, lariat-intron intermediate, lariat intron, pre-mRNA, mRNA, and 5′ exon, and the positions of RNase H cleavage products are shown. Because digestion products also arise from unspliced pre-mRNA, RNase H cleavage was determined by comparing mRNA band intensities with the no-oligo control (U) and reported as % cleaved at the bottom of each lane. (C) Same as B but with spliceosomes assembled on a pre-mRNA substrate with a 3′ ss mutation.

FIGURE 3.

UV crosslinking of proteins to exons in P complex. (A) Schematic of mRNA produced by a short 3′ exon (dark gray) pre-mRNA containing a single ³²P (marked by an asterisk) at indicated nucleotide positions relative to the eventual exon junction (marked by the black bar). The labels at the same positions were also included in a pre-mRNA with a longer 3′ exon (light gray) and mutant 3′ ss. For the +1 label, a 4-thio-uridine residue (marked by a black dot) was also included, and the mutant 3′ ss substrate also had a short 3′ exon. (B) SDS-PAGE of proteins crosslinked near the indicated single ³²P label by UV irradiation following in vitro splicing with the short 3′ exon pre-mRNA (P) or mutant 3′ ss pre-mRNA (C). As a control for proteins that bind in the absence of splicing, the labeled pre-mRNA was also incubated in splicing reactions lacking ATP at 4°C, followed by UV crosslinking (H). Positions of molecular weight marker proteins are indicated. Crosslinked protein bands detected only with the short 3′ exon pre-mRNA that accumulates P complex are marked by an asterisk (*).

FIGURE 4.

Affinity purification of P complex. Affinity purification was carried out using MS2-MBP tagged pre-mRNA substrates labeled as in Figure 1. (A) Denaturing PAGE analysis of RNA from in vitro splicing reaction time points (0′, 60′), following RNase H-mediated cleavage of substrate that did not incorporate into splicing complexes (indicated by *) as detailed in Jurica et al. (2002) (80′), the material loaded onto amylose affinity column (load), or eluted from the affinity column with maltose (elution). (B) Denaturing PAGE analysis of RNA extracted from purified spliceosome complexes, which are visualized by SYBRGold staining. RNA from nuclear extract (NE) contains U2, U1, U4, U5, and U6 snRNAs as labeled. (• indicates tRNA in the extract and * indicates pre-mRNA digestion product.) RNA from purified C complex (C), mRNP complex (M), and P complex (P) assembled on the three pre-mRNA substrates shown in panel A, respectively. The positions of cleaved 5′ exon and short mRNA that copurify are noted.

FIGURE 5.

Western analysis confirms enrichment of SF3B proteins and decrease of DHX35 in P complex. For Western analysis, equal amounts of purified C and P complexes were separated by denaturing PAGE, transferred to nitrocellulose, and the same blot was probed with the antibodies indicated on the left.

FIGURE 6.

P and C complex have similar global structures that are stabilized by chemical crosslinking. (A) Transmission EM micrograph of negatively stained P complexes after affinity purification. White scale bar is 100 nm. (B) Two class-averaged views of C and P complex preferred orientations. (C) Comparisons of fixed and unfixed splicing complexes. “C native” is from Golas et al. (2010). White arrows point out the location of a density that is present only in fixed samples.