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Evidence for a group II intron–like catalytic triplex in the spliceosome

Nature Structural & Molecular Biology volume 21, pages 464–471 (2014)Cite this article

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Abstract

To catalyze pre-mRNA splicing, U6 small nuclear RNA positions two metals that interact directly with the scissile phosphates. U6 metal ligands correspond stereospecifically to metal ligands within the catalytic domain V of a group II self-splicing intron. Domain V ligands are organized by base-triple interactions, which also juxtapose the 3′ splice site with the catalytic metals. However, in the spliceosome, the mechanism for organizing catalytic metals and recruiting the substrate has remained unclear. Here we show by genetics, cross-linking and biochemistry in yeast that analogous triples form in U6 and promote catalytic-metal binding and both chemical steps of splicing. Because the triples include an element that defines the 5′ splice site, they also provide a mechanism for juxtaposing the pre-mRNA substrate with the catalytic metals. Our data indicate that U6 adopts a group II intron–like tertiary conformation to catalyze splicing.

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Figure 1: Base-triple interactions in the group II intron catalytic core and their proposed counterparts in the spliceosome.
Figure 2: Genetic evidence for a base-triple interaction between U80 and C61 in the U6 snRNA.
Figure 3: Genetic evidence for base-triple interactions between the AGC triad and the ACAGAGA 5′ splice site–binding region of the U6 snRNA.
Figure 4: The U6 triplex forms in vitro.
Figure 5: The NTC promotes formation of the U6 triplex.
Figure 6: The U6 triplex is present at the branching and exon-ligation stages of splicing.
Figure 7: The U6 triplex promotes catalytic-metal binding during branching.
Figure 8: The U6 triplex promotes exon ligation in vitro.

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Acknowledgements

We thank S.-C. Cheng (Academia Sinica) for strains; C. Guthrie (University of California, San Francisco) and B. Schwer (Weill Cornell Medical College) for antibodies; D. Semlow (Staley laboratory) for recombinant proteins and Cy3-labeled UBC4 pre-mRNA; and members of the Staley and Piccirilli laboratories for critical discussions and comments on the manuscript. M.A.M. was supported by US National Institutes of Health training grant (T32 GM007183); this work was funded by a grant from the US National Institutes of Health (R01GM088656) to J.P.S. and J.A.P.

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Author notes

  1. Melissa A Mefford

    Present address: Present address: Department of Biology, Johns Hopkins University, Baltimore, Maryland, USA.,

  2. Sebastian M Fica and Melissa A Mefford: These authors contributed equally to this work.

Authors and Affiliations

  1. Graduate Program in Cell and Molecular Biology, University of Chicago, Chicago, Illinois, USA

    Sebastian M Fica

  2. Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois, USA

    Sebastian M Fica & Jonathan P Staley

  3. Committee on Genetics Genomics and Systems Biology, University of Chicago, Chicago, Illinois, USA

    Melissa A Mefford

  4. Department of Chemistry, University of Chicago, Chicago, Illinois, USA

    Joseph A Piccirilli

  5. Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA

    Joseph A Piccirilli

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Contributions

S.M.F., M.A.M., J.A.P. and J.P.S. designed the study; M.A.M. performed the in vivo genetics; S.M.F. performed the in vitro cross-linking and biochemistry experiments as well as experiments in Supplementary Figure 7, which were initiated by M.A.M.; and S.M.F. and J.P.S. wrote the paper with input from M.A.M. and J.A.P.

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Integrated supplementary information

Supplementary Figure 1 Further evidence that mutations in predicted base-triple partners suppress only AGC triad mutations in the same plane of the predicted triplex.

(a-d), Spot assays showing growth on selective media of equivalent numbers of yeast cells containing combinations of alleles at U80 and G60 (a), G52 and A59 (b), A53 and A59 (c), or A53 and G60 (d). The allele combinations for the U2/U6 helix Ib base pair mutated in each case are indicated above each panel, and the allele of the predicted base triple partner is shown to the left of each panel. Note that at each triad position, at least one mutation shown here as not suppressed by an out-of-the-plane mutation in the predicted triplex is nevertheless suppressed by an in-the-plane mutation of the predicted triplex.

Supplementary Figure 2 Further characterization of U6 4SU80 cross-links.

a, Denaturing PAGE analysis of ACT1 pre-mRNA splicing by extracts reconstituted with the indicated synthetic U6 snRNA. Dep., depletion; Rec., reconstitution. b-c, Denaturing PAGE analysis of U6-4SU80 recovered from in vitro splicing reactions after UV irradiation. Reactions were performed in the presence of unlabeled ACT1 pre-mRNA. Note that X2 occurs more efficiently in buffer alone, whereas X1 and X3 require splicing extract. d, Denaturing PAGE analysis of RNA products following P1 nuclease digestion of gel-purified U6, X1, and X2. Note that X2 digests to mononucleotides and thus does not involve an interaction between U80 and G52. e, Denaturing PAGE analysis of RNaseH digestion of gel-purified X1. Where indicated, DNA oligonucleotides complementary to U2 or U4 snRNA were used. Note that only the oligonucleotide complementary to U4 can direct RNaseH cleavage of X3, demonstrating that X3 involves an interaction between U80 and nucleotides in the U4 snRNA. f-g, Denaturing PAGE analysis of RNA products following analytic digestion of gel-purified U6, X1, and X2. Purified RNAs were digested as indicated (g). A diagram of the expected products in each case is also shown (f). The positions of the inferred products from the analytical digestions are indicated on the sides of the gel; * indicates unincorporated 32pppA used for 5' end labeling. Note that, A32p migrates similarly in lanes 7 and 8 to how 32pUp migrates in lane 9.

Supplementary Figure 3 The X1 cross-link can be detected in the presence of several dominant-negative ATPases.

a, Denaturing PAGE analysis of ACT1 premRNA splicing in the presence of the indicated recombinant proteins. Split reactions were set up with extracts used in b in the presence of radiolabeled ACT1 pre-mRNA. b, Denaturing PAGE analysis of U6-4SU80 recovered from in vitro splicing reactions after UV irradiation. Splicing was performed in the presence of unlabeled ACT1 pre-mRNA. Error bars represent s.d. of three independent experiments.

Supplementary Figure 4 Cwc2 promotes formation of the stacking interaction between U6 U80 and U6 G52.

a, Denaturing PAGE analysis of splicing of ACT1 premRNA in mock-depleted (M) and Cwc2p-depleted extracts (D) and reconstituted with U6-4SU80. b, Denaturing PAGE analysis of U6-4SU80 recovered from in vitro splicing reactions after UV irradiation. Splicing was performed in the presence of unlabeled ACT1 pre-mRNA and Prp2p K252A was added in order to stall spliceosomes prior to the final stage of spliceosome activation and to increase the signal for the X1 crosslink. c, Quantification of crosslinking efficiency. Values were normalized to the efficiency observed for the mock-depleted extract. In a and c error bars denote s.d. of three technical replicates. Note that depletion of Cwc2p reduced by 2.5-fold the efficiency of X1 and addition of rCwc2 restored crosslinking efficiency to a level similar to that observed for the mock-depleted extract and in proportion to the levels Cwc2p restored splicing in the depleted extract (compare with a).

Supplementary Figure 5 The U6 triplex is present during branching and exon ligation: controls for the specific association of the X1 cross-link with Prp16p.

a, Denaturing PAGE analysis of U6-4SU80 recovered after UV irradiation and immunoprecipitation with Prp16p of the indicated complex isolated from glycerol gradient fractions illustrated in Fig. 6b. PAS, protein A-sepharose. Lower panel shows quantification of IP efficiency. Note that the anti-Prp16 antibody immunoprecipitated U6 and X1 5-fold above background binding to beads. b, Denaturing PAGE analysis of U6-4SU80 recovered after UV irradiation and immunoprecipitation with Cwc25-HA of the indicated complexes isolated from glycerol gradient fractions illustrated in Fig. 6b. A representative gel for the input and immunoprecipitated material (αHA) from glycerol gradient-fractionated spliceosomes (GG s'some) is shown. The lower panel shows the UBC4 substrate present in the fractions used for immunoprecipitation, detected by Cy3 channel, as well as quantification of X1 immunoprecipitation efficiency relative to the input. The X1 immunoprecipitation efficiency for the Bact peak (lane 4) was further normalized to that for the B*(Prp16) peak (lane 3), which was set to 1. Note that X1 is enriched in the Cwc25p immunoprecipitate when spliceosomes have undergone Prp2-dependent activation (lane 3), and X1 is de-enriched in the immunoprecipitate from splicesomes stalled before the Prp2 step (lane 4). Error bars represent s.d. of three technical replicates.

Supplementary Figure 6 U6 triplex mutations do not affect branching of the 3'-OPO substrate.

a, Denaturing PAGE analysis of splicing of UBC4 pre-mRNA by affinitypurified spliceosomes reconstituted with the indicated U6 variants. No dep., no depletion; no rec., no U6 reconstitution. (b-c) Quantification of branching (b) and exon ligation (c) efficiencies, normalized to wild-type. Spliceosomes from extracts reconstituted with the indicated U6 variants were assembled on the UBC4 3ʹO-PO substrate, affinity-purified via Prp19p and incubated in buffer PK (pH 7.0) with 1 mM MgCl2. Splicing efficiencies were calculated for spliceosomes following affinity purification and subsequent incubation. Error bars represent s.d. of three technical replicates; inc., incubation.

Supplementary Figure 7 Further evidence that the U6 triplex functions during both steps of splicing.

a, Denaturing PAGE analysis of splicing of ACT1 pre-mRNA in extracts reconstituted with the indicated U6 variants. No dep., no depletion; no rec., no U6 reconstitution. b, Quantififcation of exon ligation for the indicated U6 variants, normalized to wild-type U6; exon ligation was calculated as mRNA/lariat intermediate (ref. S2). The efficiency of branching was within 10% of wild-type for all U6 variants (quantification not shown). Error bars represent s.d. of four independent experiments; **, denotes statistical significance of the difference between the exon ligation efficiencies of U6-G60U and U6-G60U/U6-G52U (p=0.0004, paired, 1-tailed, t-test). c, Spot assays showing growth on selective media of equivalent numbers of yeast cells expressing wild-type PRP16 or prp16-302 and containing the indicated U6 variants. Two 6-fold serial dilutions are shown. d, Spot assays showing ACT-CUP1 reporter-dependent growth of yeast containing the indicated U6 and ACT-CUP1 variants on media containing various concentrations of Cu2+. e, Denaturing PAGE analysis of splicing of ACT1 brG pre-mRNA by extracts reconstituted with the indicated U6 variants. Spliceosomes from extracts reconstituted with the indicated U6 variants were assembled on an ACT1 pre-mRNA bearing a guanosine at the branch site. To increase signal for the excised intron, spliceosomes were affinity-purified via Prp19p. The exon ligation efficiency was quantified without further incubation and is shown in the right panel as EI/(EI+LI), where EI is the excised intron and LI the lariat intermediate. Error bars represent s.d. of three technical replicates.

Supplementary Figure 8 Original images used to prepare display items for the main figures.

a, Full gel used for Fig. 4d. b, Full gel used for Fig. 4b. c, Full gel used for Fig. 5a. d, Full gel used for Fig. 5b. e, Full gel used for Fig. 5c. f, Full gel used for upper panels in Fig. 6c and 6d. Note that the image shown in Fig. 6d is flipped along the vertical axis, relative to the full gel shown here. g, Full gel used for lower panel in Fig. 6c. h, Full gel used for lower panel in Fig. 6d. i, Full gel used for Fig. 8b.

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Fica, S., Mefford, M., Piccirilli, J. et al. Evidence for a group II intron–like catalytic triplex in the spliceosome. Nat Struct Mol Biol 21, 464–471 (2014). https://doi.org/10.1038/nsmb.2815

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