The architecture of the spliceosomal U4/U6.U5 tri-snRNP

Abstract

U4/U6.U5 tri-snRNP is a 1.5-megadalton pre-assembled spliceosomal complex comprising U5 small nuclear RNA (snRNA), extensively base-paired U4/U6 snRNAs and more than 30 proteins, including the key components Prp8, Brr2 and Snu114. The tri-snRNP combines with a precursor messenger RNA substrate bound to U1 and U2 small nuclear ribonucleoprotein particles (snRNPs), and transforms into a catalytically active spliceosome after extensive compositional and conformational changes triggered by unwinding of the U4 and U6 (U4/U6) snRNAs. Here we use cryo-electron microscopy single-particle reconstruction of Saccharomyces cerevisiae tri-snRNP at 5.9 Å resolution to reveal the essentially complete organization of its RNA and protein components. The single-stranded region of U4 snRNA between its 3′ stem–loop and the U4/U6 snRNA stem I is loaded into the Brr2 helicase active site ready for unwinding. Snu114 and the amino-terminal domain of Prp8 position U5 snRNA to insert its loop I, which aligns the exons for splicing, into the Prp8 active site cavity. The structure provides crucial insights into the activation process and the active site of the spliceosome.

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Figure 1: Overview of the U4/U6.U5 tri-snRNP structure with its protein and RNA components modelled into cryo-EM density.
Figure 2: Prp8 in tri-snRNP.
Figure 3: The snRNA components of U4/U6.U5 tri-snRNP.
Figure 4: Structure of Snu114 in tri-snRNP.
Figure 5: Brr2 mode of unwinding.
Figure 6: Insights into activation mechanism and the active site of the spliceosome.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Data deposits

3D cryo-EM density maps of yeast U4/U6.U5 tri-snRNP have been deposited with the Electron Microscopy Database under accession number EMD-2966.

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Acknowledgements

We thank S. Chen, G. McMullan, J. Grimmett and T. Darling for smooth running of the EM and computing facilities; P. da Fonseca, N. Unwin, I. Sanchez Fernandez, A. Amunts, P. Emsley, G. Murshudov and A. Brown for advice; A. Easter and L. Passmore for reagents; M. Skehel for mass spectrometry; and J. Li, Y. Kondo and the members of the spliceosome group for help and advice throughout the project. We are grateful to R. Henderson, D. Barford, S. Fica, P.-C. Lin and L. Strittmatter for critical reading of the manuscript. We thank V. Ramakrishnan, J. Löwe and R. Henderson for their continuing support and encouragements. T.H.D.N. was supported in part by a Herchel Smith Research Studentship. X.-c.B. was supported by a European Union Marie Curie Fellowship. The project was supported by the Medical Research Council (MC_U105184330 to K.N. and MC_UP_A025_1013 to S.H.W.S.).

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Authors

Contributions

T.H.D.N. developed the purification procedure for yeast tri-snRNP, prepared EM grids, collected all EM images, processed data, calculated the maps and built and fitted most of the components into the map. W.P.G. built most of the unknown components and made essential contributions to sequence analysis, homology modelling and model fitting. X.-c.B. helped T.H.D.N. with image processing and map calculation. C.G.S. guided T.H.D.N. with EM sample preparation and data collection. A.J.N. produced the Brr2 TAPS-tagged strain and contributed to the project through his knowledge of yeast spliceosome. T.H.D.N. and W.P.G prepared all illustrations. T.H.D.N prepared the video. S.H.W.S. carried out multi-body refinement and oversaw the EM analysis. K.N. initiated and orchestrated the project. T.H.D.N., W.P.G., A.J.N. and K.N. interpreted the results and wrote the paper with crucial contribution from all other authors.

Corresponding authors

Correspondence to Thi Hoang Duong Nguyen or Kiyoshi Nagai.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 U4/U6.U5 tri-snRNP sample used for this study.

a, Coomassie-blue-stained SDS–PAGE gel showing protein composition of the purified tri-snRNP. U5-, U4/U6- and tri-snRNP-specific proteins are labelled in blue, red and teal, respectively. Sm proteins present in both U5 and U4/U6 are in black. b, Toluidine-blue-stained denaturing acrylamide (9%) gel showing RNA compositions. c, Electron cryo-micrograph of tri-snRNP where the carbon-coated grid was discharged in N-amylamine. d, e, Reference-free two-dimensional class averages of a data set collected on a grid discharged in air and N-amylamine, respectively.

Extended Data Figure 2 Classification and refinement procedures used in this study.

A total of 367,327 particles were subjected to reference-free 2D classification. A subset of 347,241 particles from good 2D classes was selected for 3D classification using an initial model obtained from SIMPLE-PRIME53, which was low-pass filtered to 60 Å. The data were divided into four 3D classes, two of which (a total of 179,079 particles) showed better features and were combined for refinement. This resulted in a 7.6 Å reconstruction. To further improve the reconstruction, these particles were subjected to beam-induced motion correction (particle polishing)24. Refinement of these polished particles with a soft mask around the rigid part of the map (as indicated by the red envelope) yielded a 5.9 Å reconstruction while refinement with a mask around the whole map yielded a 6.4 Å reconstruction. The polished particles were also subject to further 3D classification with a finer angular sampling of 1.8°. The most populated class (47,674 particles), which also has the best rotational accuracy, was refined with a soft mask around the whole density. This resulted in a 7.0 Å reconstruction. In this study, the 5.9 Å reconstruction was used for subsequent biological interpretation. All steps were performed in RELION22 unless otherwise stated.

Extended Data Figure 3 CryoEM maps and tilt-pair validation.

a, CryoEM density of the whole tri-snRNP at 5.9 Å resolution by ‘gold standard’ Fourier shell correlation (FSC) of 0.143 criterion at two different contour levels. The high contour map (gold) shows well-resolved densities for protein and RNA helices and flat densities for β-sheets. The low contour map (silver) shows densities for the more flexible head and arm. The map was sharpened by a B-factor of −214 Å2 and low-pass filtered to 5.9 Å as determined by RELION. b, The unsharpened full map of tri-snRNP. c, The map resulting from multi-body refinement, in which tri-snRNP is divided into four parts: the head, body, arm and foot. This resulted in better density for the arm domain (indicated by red circles), which is at 20 Å resolution. d, Tilt-pair validation plot for tri-snRNP. This was obtained from 1,196 particles from 32 micrograph pairs, imaged at 0° and 10° tilt angles. The position of each dot represents the direction and the amount of tilting for a particle pair in polar coordinates. Blue dots correspond to in-plane tilt transformations; red and purple dots correspond to out-of-plane tilt transformations. Blue dots cluster in the same region of the plot at a tilt angle of approximately 10° as indicated by the red circle.

Extended Data Figure 4 Resolution estimation of tri-snRNP map.

a, Local resolution of the tri-snRNP map estimated by ResMap using the colour scheme shown in panel c. b, Local resolution of the tri-snRNP map calculated by ‘gold-standard’ FSC. For each component of the map that we modelled protein/RNA components, a soft mask (with a 30-pixel soft edge) surrounding the region of interest was prepared and used for FSC calculations. Convolution effects of the masks on the FSC curves were corrected using high-resolution noise substitution55. Resolution was estimated at FSC = 0.143. Local resolution for the unmodelled region of the map (in red) was not estimated. c, Local resolution of model versus map. The map of each modelled component was extracted from the map using a soft mask (with a 5-pixel soft edge) surrounding the component. The model was converted into density by EMAN57. FSC of model versus map was calculated using Xmipp56. The map is coloured according to resolution estimates based on a FSC threshold of 0.25. The lower resolution estimates from the FSC of model versus map compared to the estimates from ResMap and the gold-standard FSCs are explained by the nature of our models. Because of the limited resolution of our map, we did not perform full atomic refinement, but placed known crystal structures and homology models as rigid bodies in the map. d, Gold-standard FSC curves for the whole tri-snRNP map and some of its components calculated as described in b. e, FSC curves of model versus map for the whole model and some of the components. f, The full tri-snRNP map in which portions of the structure produced from crystal structures, homology modelling and de novo building or unmodelled are coloured as indicated.

Extended Data Figure 5 Fitting of protein components into tri-snRNP map.

a, Prp8(885–2,413) crystal structure10 (PDB 4I43, green) and additional helices built de novo assigned to the N terminus of Prp8 (blue). b, Brr2–Jab1/MPN complex31 (PDB 4BGD). c, Snu114 homology model based on EF2 (ref. 26). d, The Prp6 TPR motifs built into the tri-snRNP map. e, U5 Sm proteins (grey) with Sm site (blue) based on the human U4 Sm structure (PDB 4WZJ). f, Dib1 (ref. 29) (PDB 1QGV). g, (i) Prp31. (ii), Comparison between the crystal structure of human Prp31(78–333) (ref. 33) (PDB 2OZB, grey) and that in tri-snRNP (yellow and blue). The coiled-coil domain (yellow) rotates by 60° in tri-snRNP with respect to the Nop domain (grey). Additional helices (blue) that extend from the N and C termini were built. h, U4 Sm proteins with part of U4 snRNA (blue) based on the human U4 Sm structure. i, Prp3 model. The ferredoxin-like domain was obtained from homology modelling while the extra helices were built de novo. j, Prp4 WD40 homology model with the extra helices built de novo. k, Snu13 (ref. 64) (PDB 2ALE). l, U6 LSm proteins67 (PDB 4M77).

Extended Data Figure 6 Fitting of the RNA components in tri-snRNP map.

a, c, The sequences and predicted secondary structures of U4/U6 snRNA and the long version of U5 snRNA, respectively. b, d, The maps of the fitted parts of U4/U6 snRNA and U5 snRNA, respectively. Unmodelled density assigned to U5 snRNA is also shown in d.

Extended Data Figure 7 Sequence alignment of yeast and human Snu114 with yeast and human elongation factor 2 (EF-2).

The secondary structures of our homology model for yeast Snu114 and the yeast EF-2 (ref. 26) (PDB 1N0V) are shown on the top and bottom of the alignment, respectively. Important sequence elements are also shown. The greyscale shading indicates the level of sequence conservation. A higher level of conservation is shown in a darker shade.

Extended Data Figure 8 The effect of ATP on Brr2-TAPS purified tri-snRNP.

a, Ethidium-bromide-stained native agarose gel (0.5%) showing the effects of ATP addition to Brr2-TAPS purified tri-snRNP used in this study. Upon ATP addition either without or with GTP/GDP, tri-snRNP fell apart (lanes 1–4). Under the same conditions, the addition of ADP or the non-hydrolysable ATP-analogue, AMPPNP, had no effects on the complex (lanes 5, 6). b, c, The effect of ATP addition observed by negative stain microscopy. When ATP was not present, tri-snRNP particles could be observed. When ATP was added to the sample before grid preparations, tri-snRNP particles fell apart as observed by many small components on the micrograph rather than tri-snRNP particles. d, Tri-snRNP model where U4/U6 snRNP proteins are not shown. In tri-snRNP, Brr2–Prp8Jab complex is loosely associated to the remaining U5 snRNP components including Prp8large, Prp8RNaseH, Prp8Nterm, Snu114, Dib1, U5 Sm proteins and U5 snRNA. After U4/U6 snRNA unwinding by Brr2, Brr2–Prp8Jab could be repositioned within the spliceosome. e, A schematic showing the arrangement of tri-snRNP protein and RNA components.

Extended Data Table 1 Components and modelling of yeast U4/U6.U5 tri-snRNP
Extended Data Table 2 U4-cs1 suppressors

Supplementary information

The architecture of the spliceosomal U4/U6.U5 tri-snRNP

The video sequences showing the cryoEM density at two different contour levels; tri-snRNP map with all modeled components; fitting of available crystal structures into the cryoEM density: Brr2-Jab1/MPN (Prp8) complex31, Prp8 RNase H and large domains10, U4 and U5 Sm core domains65, Lsm core domain67 fitted into the multi-body map, Snu13 (ref. 64), human Prp31 (ref. 33) with remodeling, human Dib1 (ref. 29); fitting of homology models: Snu114 based translation factor EF2 (ref. 26), WD40 domain of Prp4, ferredoxin-like domain of Prp3 (ref. 36), TPR domain of Prp6; fitting of double helical RNA of the U4/U6 snRNA duplex and U5 snRNA; fitting of α-helices attributed to the N-terminal domain Prp8, Prp3 and Prp4; near complete pseudo-atomic structure of the yeast U4/U6.U5 tri-snRNP. (MOV 42926 kb)

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Nguyen, T., Galej, W., Bai, X. et al. The architecture of the spliceosomal U4/U6.U5 tri-snRNP. Nature 523, 47–52 (2015). https://doi.org/10.1038/nature14548

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