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Structure of a pre-catalytic spliceosome

Nature volume 546, pages 617–621 (2017)Cite this article

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Abstract

Intron removal requires assembly of the spliceosome on precursor mRNA (pre-mRNA) and extensive remodelling to form the spliceosome’s catalytic centre. Here we report the cryo-electron microscopy structure of the yeast Saccharomyces cerevisiae pre-catalytic B complex spliceosome at near-atomic resolution. The mobile U2 small nuclear ribonucleoprotein particle (snRNP) associates with U4/U6.U5 tri-snRNP through the U2/U6 helix II and an interface between U4/U6 di-snRNP and the U2 snRNP SF3b-containing domain, which also transiently contacts the helicase Brr2. The 3′ region of the U2 snRNP is flexibly attached to the SF3b-containing domain and protrudes over the concave surface of tri-snRNP, where the U1 snRNP may reside before its release from the pre-mRNA 5′ splice site. The U6 ACAGAGA sequence forms a hairpin that weakly tethers the 5′ splice site. The B complex proteins Prp38, Snu23 and Spp381 bind the Prp8 N-terminal domain and stabilize U6 ACAGAGA stem–pre-mRNA and Brr2–U4 small nuclear RNA interactions. These results provide important insights into the events leading to active site formation.

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Figure 1: B complex structure at near-atomic resolution.
Figure 2: U2 snRNP architecture and interactions with the intron.
Figure 3: U2 snRNP and tri-snRNP interfaces.
Figure 4: B complex proteins stabilize Brr2 and U6 snRNA.
Figure 5: Changes in the RNA network during spliceosome activation.
Figure 6: Model for spliceosome activation.

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Acknowledgements

We thank C. Savva, S. Chen, G. Cannone, K. R. Vinothkumar, G. McMullan, J. Grimmett and T. Darling for maintaining electron microscopy and computing facilities; the mass spectrometry facility for protein identification, and A. Newman, L. Strittmatter, S. Fica, M. Wilkinson and C. Norman for help and critical reading of the manuscript. We thank J. Löwe, V. Ramakrishnan, D. Barford and R. Henderson for their continuing support. The project was supported by the Medical Research Council (MC_U105184330) and European Research Council Advanced Grant (693087 - SPLICE3D). C.P. was supported by an EMBO Long-Term Fellowship.

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  1. Clemens Plaschka and Pei-Chun Lin: These authors contributed equally to this work.

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  1. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2 0QH, UK

    Clemens Plaschka, Pei-Chun Lin & Kiyoshi Nagai

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  1. Clemens Plaschka
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Contributions

C.P. established complex preparation. C.P. and P.-C.L. performed cryo-EM structure determination and model building. C.P. refined the model. C.P., P.-C.L. and K.N. analysed the structure and wrote the manuscript. K.N. initiated and supervised the project.

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Correspondence to Clemens Plaschka, Pei-Chun Lin or Kiyoshi Nagai.

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Extended data figures and tables

Extended Data Figure 1 Biochemical characterization and cryo-EM of the B complex spliceosome.

a, Protein analysis of purified B complex (SDS–PAGE stained with Coomassie blue). U1 snRNP components and U1 snRNA are sub-stoichiometric (see b), consistent with U1 snRNP destabilization in B complex2,31. For gel source data see Supplementary Fig. 1a. b, RNA analysis of purified B complex (denaturing 9% polyacrylamide TBE gel stained with Toluidine blue). For gel source data see Supplementary Fig. 1b. c, Purified B complex is active in an in vitro splicing assay. Splicing reactions were carried out in yeast extract in the absence (lanes 1 and 2) or presence of 60 nM unlabelled competitor pre-mRNA (lane 3), prohibiting the assembly of new spliceosomes. B complex was assembled on labelled pre-mRNA and purified (see Methods), and added to yeast extract for 10 min together with 60 nM unlabelled competitor pre-mRNA, before the addition of ATP to initiate the reaction (lanes 4 and 5). Splicing reactions contained 2 mM ATP (lanes 1–5). The asterisk marks a degradation product. For gel source data see Supplementary Fig. 1c. d, Cryo-EM micrograph of B complex. Scale bar, 200 nm. e, Representative 2D class averages of B complex reveal flexibility of peripheral regions relative to the tri-snRNP body. f, Composite cryo-EM density of B complex shown in two orthogonal views. Colours indicate the respective cryo-EM densities used for modelling (B1, green; B2, yellow; B3, light green; B4, grey; B5, blue; B6, light blue; B7, magenta). The sharpened densities are shown and are aligned using overlapping regions (see Extended Data Fig. 2). The percentage of particles from the full set of 496,581 that contribute to the respective density is indicated together with the overall resolution (see Extended Data Fig. 9). g, Composite cryo-EM density of B complex superimposed on a ribbon model of the B complex structure, coloured as in Fig. 1. The B complex, excluding the U1 snRNP, has a molecular mass of 2.5 MDa of which we modelled 1.8 MDa.

Extended Data Figure 2 Three-dimensional classification of cryo-EM data.

Three-dimensional image classification of the cryo-EM dataset using the B complex negative-stain reconstruction (Methods; Extended Data Fig. 3b) as the initial reference model. The percentage of single particles contributing to each class is provided. To help visualize structural differences, 3D reconstructions of B complex are coloured according to mobile regions: SF3b (green), U2 3′ domain–SF3a (light green), helicase (cyan), body (grey), foot (navy blue), and B complex proteins (magenta). For each classification round, the type of mask and use of signal subtraction is indicated. Additionally, the type of mask, overall resolution, and use of signal subtraction is also indicated for each 3D refinement after classification. For additional details, see the Methods and Extended Data Fig. 9.

Extended Data Figure 3 Negative-stain and cryo-EM reconstructions of B complex.

a, Three-dimensional image classification of negative-stain EM data. A total of 12,296 particles were refined using the negative-stain reconstruction of the human BΔU1-complex32 (EMDB code EMD-1066) as the initial reference, and were subsequently classified. Class 2 contained most features and was used for 3D refinement. The percentage of single particles contributing to each class is provided. b, Two orthogonal views of the yeast B complex negative-stain reconstruction used as the initial reference for processing of the cryo-EM dataset. c, Gold-standard Fourier shell correlation (FSC = 0.143) of the respective B1, B2, B3, B4, B5, B6 and B7 cryo-EM single-particle reconstructions. d, Orientation distribution plot of all particles that contribute to the respective B1, B2, B3, B4, B5, B6 and B7 cryo-EM single-particle reconstructions. e, The composite B complex cryo-EM density (maps B1–B7) is shown in two orthogonal views and coloured by local resolution, as determined by ResMap64. Compare with Extended Data Fig. 1f. f, A central slice through the composite B complex cryo-EM density (maps B1–B7) is shown in two orthogonal views and coloured by local resolution, as determined by ResMap64.

Extended Data Figure 4 Details of the U2 snRNP.

a, Multiple conformations of the U2 snRNP 3′ domain–SF3a subcomplexes relative to SF3b indicate flexibility. This apparent mobility may be important for formation of the A complex, consistent with dynamic contacts of the U2 snRNA 5′ end with SF3a60 (human PRP9) and SF3b49 (human HSH49) in the isolated U2 snRNP40 that differ from U2 snRNP protein–snRNA interactions observed in the yeast B complex structure. Surface representations of the U2 3′ domain (light green), SF3a (light yellow), and SF3b (dark green) are shown. The U2 3′ domain–SF3a complex is positioned according to 3D classifications 1, 6 and 7 from round 5 (compare with Extended Data Fig. 2). A cartoon summarizes the movements. b, Representative regions of the sharpened SF3b-containing density (B1) at 3.9 Å resolution are superimposed on the refined coordinate model. The density shows side-chain features for a loop in Cus1, a β-strand in Rse1, an α-helix in Hsh155, and separation of RNA nucleotides in the U2–pre-mRNA branch helix. Colours are as in Fig. 2. c, Cryo-EM densities for SF3a are superimposed on the B complex coordinate model. The crystal structure of the Y-shaped core of SF3a (ref. 29) is superimposed on the B3 density. Prp11 ZnF, Prp9 ZnF2 and the Prp9 C terminus are superimposed on the B1 density. Structural elements of SF3a, including the Prp9 wedge helix, and disordered regions are indicated. For cryo-EM density nomenclature, see Extended Data Fig. 1f. Colours are as in Fig. 2.

Extended Data Figure 5 Flexibility of the U2 snRNP relative to tri-snRNP.

a, Multiple positions of the U2 snRNP relative to tri-snRNP. Representative classes are shown (classes 1, 6 and 8 from round 3, see Extended Data Fig. 2) that reside along a continuum of conformations (compare with Extended Data Fig. 1e). The U2 snRNP moves together with the U6 LSm ring, which is anchored via the putative Prp3 N terminus. A cartoon summarizes the movements in two orthogonal views. The location of the U2 snRNP has an apparent effect on the strength of the putative pre-mRNA 5′ exon cryo-EM density (compare Extended Data Fig. 6e). When the U2 snRNP is positioned away from the U6 ACAGAGA stem, the pre-mRNA density is weaker than when the U2 snRNP is positioned closer. This suggests how Brr2 helicase activity may perform a kinetic proofreading of the pre-mRNA 5′-exon–U6 snRNA interaction: When the U2 snRNP is close to the tri-snRNP, activation may occur normally (productive activation). However, when the U2 snRNP is positioned further away and the 5′ exon is not tethered, Brr2 activity may instead lead to dissociation of the U2 snRNP from the tri-snRNP (non-productive activation)12. b, The Prp3 model is superimposed on B1 (putative N-terminal region), B2 (helix α4) and B4 (C-terminal region) cryo-EM densities. The Prp3 ferredoxin-like fold (FER) and secondary structure elements are labelled. The black arrowhead indicates the region of Prp3 helix α4 that bends with different U2 snRNP positions (see a). For cryo-EM density nomenclature, see Extended Data Fig. 1f.

Extended Data Figure 6 Details of B complex proteins and U6 snRNA.

a, Fit of the Spp381 model to B6 (helices α1–α2) and B7 (helices α3–α4) cryo-EM densities. Helix α4 was modelled into weak density (B7) based on homology to the human and Chaetomium thermophilum crystal structures46. For cryo-EM density nomenclature, see Extended Data Fig. 1f. b, Fit of the Prp38 model to the B7 cryo-EM density. Helix α5 is shown below, revealing side-chain features in the density. c, Fit of the Snu23 model to B6 (helix α1) and B7 (remainder of Snu23) cryo-EM densities. Helix α2 is shown below, revealing side-chain features in the density. d, Composite cryo-EM density and fit of the U6 snRNA model. The B1, B2, B4, B5 and B7 densities are shown without (left) and with (right) the U6 snRNA model superimposed. U6 elements and the site of interaction with the U5 snRNA loop 1 are indicated. e, A weak density for pre-mRNA connects from the U6 snRNA ACAGAGA stem to the U2 SF3b-bound intron. The connecting density (map B2) is shown at intermediate (grey, threshold of 0.023) and low (light blue, threshold of 0.0173) thresholds. The U6 snRNA densities are shown as in d. The register of pre-mRNA near the U6 ACAGAGA stem is uncertain and was tentatively modelled based on complementarity with UBC4 pre-mRNA upstream and nearest to the 5′ exon, consistent with RNA crosslinking48. According to this register, approximately 40 pre-mRNA nucleotides separate U6- and SF3b-bound regions. The bottom right panel shows the fit of the pre-mRNA–U6 snRNA ACAGAGA stem–loop model to the unsharpened B7 density. For cryo-EM density nomenclature, see Extended Data Fig. 1f. f, Snu23 binds near the nucleotide-binding pocket of the Brr2 NHC, where it may influence Brr2 activity. Brr2 (pale cyan) and Snu23 (violet) models are superimposed on the B6 cryo-EM density, coloured as the underlying proteins. The RecA-1 and RecA-2 lobes of the Brr2 NHC are labelled, and an ADP nucleotide was modelled by aligning the NHC of human BRR2 bound to ADP73 (PDB code 4KIT) on the equivalent yeast Brr2 residues. The path of the Snu23 N terminus cryo-EM density is indicated, and is near to the Brr2 nucleotide-binding pocket. g, The U6 ACAGAGA stem is chaperoned by Dib1, Prp6, Prp8 and B complex proteins. Stabilization of the U6 ACAGAGA stem may facilitate tethering of pre-mRNA at its tip, whereas the U6 ACAGAGA box is buried in the stem. Surface models of Snu23 ZnF, Prp38 N terminus, Prp6 N terminus, Dib1, Prp8L and Prp8N domains, and Brr2 are shown and reveal a network of protein–RNA contacts to maintain the U6 ACAGAGA stem. h, Comparison of human tri-snRNP25 (PDB code 3JCR) and yeast B complex (this study) reveals that the Prp8N and Prp8EN domains serve as a platform for mutually exclusive binding of Prp28 and B complex proteins. Their Prp8N binding sites overlap and the altered location of the Prp8EN domain between the two complexes provides additional interfaces for either Prp28 or B complex proteins to bind. Movements compared to B complex in Brr2 and U4/U6 di-snRNP are indicated with arrows, and may occur after A complex association. The U2 snRNP is shown in grey to highlight tri-snRNP components, which are coloured as in Fig. 1. Comparison with Bact and C* structures20,21,22,23,24 further suggests that the Prp8N and Prp8EN domains serve as a general platform for step-specific splicing factors during the splicing cycle.

Extended Data Figure 7 U5 snRNA model, location of the U1 snRNP, and RNA secondary structure diagrams.

a, An improved model for U5 snRNA. A secondary structure diagram (left) and refined coordinate model (right) are shown, and the newly determined variable stem–loop II, stem III and stem IV are labelled together with known U5 snRNA elements. The long form of U5 snRNA is shown, where the short form ends with nucleotide 179. The grey boxes indicate regions not included in the model. Lines indicate Watson–Crick base pairs, and dots indicate G–U wobble base pairs. The U5 snRNA model was prepared by M. Wilkinson. b, Putative model of the RNA interaction network in the pre-B complex. The RNA network is unchanged compared to B complex, except for the interaction of the pre-mRNA 5′-exon with U1 snRNA74. Only loop 1 of U5 snRNA is shown. Lines indicate Watson–Crick base pairs, dots G–U wobble base pairs, stars denote non-canonical base pairs, and dotted lines putative nucleotide interactions. c, Putative location of the U1 snRNP. To gain insights into U1 snRNP location relative to U2 snRNP and tri-snRNP, we combined genetic, biochemical and structural observations. The U1 snRNP may bind between the human SF3a subunit SF3A1 (yeast Prp21)75, the Prp28-binding site25, Brr2 (ref. 76), and the U6 ACAGAGA stem. In B complex, the U1 snRNP may be destabilized owing to a steric clash with Brr2, which is likely to be repositioned compared to the pre-B complex, as in the human tri-snRNP structure25. In humans, the U1 snRNP may be further destabilized by a loss of A complex-specific proteins2. Brr2 repositioning may therefore serve as a checkpoint to signal the release of U1 snRNA from pre-mRNA. d, RNA secondary structure diagrams of regions modelled in B and Bact complex spliceosomes, using UBC4 pre-mRNA. The pre-mRNA substrate in Bact (ref. 21) is a mixture of cellular pre-mRNAs and its sequence is replaced by that of UBC4. The consensus nucleotides at the 5′SS and branch point sequence in yeast are shown in bold. Only loop 1 of U5 snRNA is shown. Lines indicate Watson–Crick base pairs, dots denote G–U wobble base pairs, stars denote non-canonical base pairs, and dotted lines putative nucleotide interactions. Highlighted are the branch-point adenosine, pre-mRNA 5′ and 3′ exons, and the U6 ACAGAGA box.

Extended Data Figure 8 Compositional and conformational changes during spliceosome activation.

a, List of RNA and protein components in B and Bact complex spliceosomes. During spliceosome activation, 22 proteins join the spliceosome, whereas 24 proteins (or 41 including the U1 snRNP) and U1 and U4 snRNAs are released. The U1 snRNP is indicated by a dashed line owing to its substoichiometric binding in B complex. RES complex proteins were not modelled in B complex, however, weak density is visible at the same binding site as in Bact (refs 20, 21), consistent with mass spectrometry data (ref. 9 and data not shown). b, Movement of the Prp8 RNase H domain (Prp8RH) between B and Bact complex spliceosomes. In B complex (left) regions of Prp3, Prp6 and Snu66 (residues 148–236) contact the Prp8RH and its β-hairpin (red). The Prp8 large domain (Prp8L) is shown as a surface and subunits are coloured as in Fig. 1. An arrow indicates the movement of Prp8RH to its Bact position21 (right; PDB code 5GM6), where it is stabilized by Hsh155, Prp45 (yellow) and Cwc22 (dark violet). B and Bact models were aligned on the Prp8L. c, Movements of the Prp8 switch loop and N-terminal domain between B and Bact complex spliceosomes. In B complex (left), an unassigned peptide (orange) binds the Prp8 switch loop, stabilizing it on the Prp8L domain. The Prp8 N-terminal domain (Prp8N) is shown as a surface (magenta) and binds the U6 snRNA 5′ stem. The Prp8 endonuclease (Prp8EN) domain is labelled. Arrows indicate the movements required to transition to Bact (PDB code 5GM6). The unassigned peptide is released in Bact (right) and Cwc21 (yellow) and Cwc22 (dark violet) bind in its stead to stabilize the new position of the Prp8 switch loop and the loaded pre-mRNA 5′ exon (light orange) in the exon channel. The repositioned Prp8N completes this channel. B and Bact models were aligned on the Prp8L domain. d, Movements of U2 3′ domain–SF3a subcomplexes between B and Bact complex spliceosomes. In B complex (left) the U2 3′ domain–SF3a subcomplexes are flexibly linked to SF3b and assume several positions relative to SF3b (compare Extended Data Fig. 4a). For comparison to Bact, the model for U2 3′ domain–SF3a (light green/grey) was rigid-body fitted into a low-pass filtered cryo-EM density of yeast Bact (EMDB code EMD-4099)20. This indicated that in Bact (right), the U2 3′ domain–SF3a complex is repositioned due to binding of NTC proteins Syf1 and Clf1 (yellow arch), to avoid a steric clash. The NTC subunits Syf1 and Clf1 may thus be positioned in Bact to bind the U2 3′ domain after the release of SF3a, during conversion to B* (Extended Data Fig. 8d). This repositioning is distinct from U2 3′ domain–SF3a conformations that are sampled in B complex. B and Bact models of the U2 snRNP were aligned on SF3b subunit Rse1. e, Movements of SF3b subunit Hsh155 and pre-mRNA between B and Bact complex spliceosomes. B and Bact (PDB code 5GM6) models were aligned on Hsh155, revealing small conformational changes in Hsh155 HEAT repeats, possibly owing to extended interaction of Hsh155 with Prp8, Snu17 and Prp45 (grey arches) in Bact. The U2–pre-mRNA branch helix is bound in the same manner in B and Bact complex spliceosomes. However, nucleotides downstream of the branch point (magenta) are bound differently, possibly due to movements in Hsh155. f, Structural differences between B and Bact complex spliceosomes suggest a model for activation. The ATP-dependent helicase Brr2 is positioned in B complex (left) and unwinds the U4/U6 duplex to release U4 snRNA and U4/U6 and B complex proteins (middle left). Their removal enables movements in Brr2, U2 and U5 snRNPs, and U6 snRNA to facilitate the loading of pre-mRNA, and formation of the U2/U6 catalytic centre (middle right). This intermediate is subsequently stabilized by NTC, NTR, splicing factors and Bact complex proteins to form Bact (ref. 21) (right; PDB code 5GM6). U5 snRNP (blue; U5 snRNA, light grey), U4/U6 di-snRNP (light yellow; U4 snRNA, yellow; U6 snRNA, red), U2 snRNP (U2 3′ domain, light green; SF3a, olive; SF3b, dark green), B complex proteins (shades of magenta), NTC, NTR and splicing factors (light yellow), and Bact complex proteins (shades of salmon) are indicated. The Bact position of U2 snRNP 3′ domain–SF3a was modelled as in Fig. 5. B and Bact spliceosomes were aligned on their Prp8L domain. Spliceosome activation intermediates are modelled.

Extended Data Figure 9 Data collection, refinement statistics and structure validation.

a, Cryo-EM data collection and refinement statistics of the B complex structure. Different regions of the composite B complex structure were refined into B1, B4, B5, B6 and B7 maps as described (see Methods). b, FSC between local cryo-EM map regions and corresponding regions of the refined coordinate models. Note that the FSC curve for the B-specific proteins correlates with the local resolution in this sub-region of the B7 density (4.0–5.0 Å; Extended Data Fig. 3e), below the nominal resolution (4.0 Å).

Extended Data Table 1 Summary of components modelled into B complex cryo-EM densities
Full size table

Supplementary information

Supplementary Figure

This file contains Supplementary Figure 1, comprising source data for protein and RNA gels. Supplementary Figure 1a, b, and c are the uncropped images for (a) Extended Data Fig. 1a, (b), Extended Data Fig. 1b, and (c) Extended Data Fig. c. (PDF 555 kb)

Supplementary Data

This file contains a pymol session of the B complex (PDB coordinate file: 5NRL). Colours as in Figure 1. (ZIP 5224 kb)

Structure of a pre-catalytic spliceosome

This video shows a 360 degree rotation of the B complex (PDB coordinate file: 5NRL). Colours as in Figure 1. (MOV 28942 kb)

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Plaschka, C., Lin, PC. & Nagai, K. Structure of a pre-catalytic spliceosome. Nature 546, 617–621 (2017). https://doi.org/10.1038/nature22799

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