Abstract
Selection of the pre-mRNA branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is crucial to prespliceosome (A complex) assembly. The RNA helicase PRP5 proofreads BS selection but the underlying mechanism remains unclear. Here we report the atomic structures of two sequential complexes leading to prespliceosome assembly: human 17S U2 snRNP and a cross-exon pre-A complex. PRP5 is anchored on 17S U2 snRNP mainly through occupation of the RNA path of SF3B1 by an acidic loop of PRP5; the helicase domain of PRP5 associates with U2 snRNA; the BS-interacting stem-loop (BSL) of U2 snRNA is shielded by TAT-SF1, unable to engage the BS. In the pre-A complex, an initial U2–BS duplex is formed; the translocated helicase domain of PRP5 stays with U2 snRNA and the acidic loop still occupies the RNA path. The pre-A conformation is specifically stabilized by the splicing factors SF1, DNAJC8 and SF3A2. Cancer-derived mutations in SF3B1 damage its association with PRP5, compromising BS proofreading. Together, these findings reveal key insights into prespliceosome assembly and BS selection or proofreading by PRP5.
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Data availability
The atomic coordinates for human 17S U2 snRNP and the pre-A complex have been deposited in the Protein Data Bank (PDB) under accession codes PDB 7EVO and PDB 7VPX, respectively. The EM maps of 17S U2 snRNP core region, PRP5-α1, PRP5-α2, TAT-SF1 and BSL have been deposited in the EMDB with accession codes EMD-31334, EMD-31335, EMD-31336, EMD-31337 and EMD-31338, respectively. The EM map of the pre-A complex core region, DNAJC8 region and U1 snRNP region have been deposited in EMDB with accession codes EMD-32074, EMD-32075 and EMD-32076, respectively. The accession number for the RNA sequencing data reported in this paper is from the Gene Expression Omnibus (GEO GSE185713). Source data are provided with this paper.
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Acknowledgements
We thank the Cryo-EM Facility, the Computing Center, the Crystallography Facility and the Mass Spectrometry & Metabolomics Core Facility of Westlake University for technical support. We thank N. A. Larsen from H3 Biomedicine for providing SSA. This work was supported by funds from the National Natural Science Foundation of China (31930059 to Y.S.), the China Postdoctoral Science Foundation (2020M671806 to X. Zhang; 2021M692888 to X. Zhan), the National Postdoctoral Program for Innovative Talents of China (BX20200305 to X. Zhang; BX2021268 to X. Zhan) and Start-up funds from Westlake University (to Y.S.).
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Y.S. conceived the project. X. Zhang, T.B. and F.Y. designed and performed the experiments. X. Zhang prepared cryo-EM samples and collected the EM data. X. Zhan, X. Zhang, Y.L. and Z.X. processed the EM data, calculated the EM maps, and built the atomic models. F.Y., R.F., P.L. and Q.Z. performed RNA-seq data analysis. Y.S. and X. Zhang wrote the manuscript with input from all authors.
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Extended data
Extended Data Fig. 1 Purification and cryo-EM reconstruction of human 17S U2 snRNP.
a, Only U2 snRNA is detectable in the purified human 17S U2 snRNP as shown in this denaturing PAGE gel. b, Analysis of the purified human 17S U2 snRNP on a silver-stained SDS-PAGE gel. The protein components were identified by mass spectrometry. c, A representative cryo-EM micrograph (upper panel) and representative 2D class averages (lower panel) of the human 17S U2 snRNP sample. The experiments were repeated independently for more than three times with similar results. d, A flow chart diagram of cryo-EM data processing for human 17S U2 snRNP. Please refer to Methods for details. e, The final reconstructions for the masked core (red), PRP5-α1 (blue), PRP5-α2 (yellow), TAT-SF1(magenta) and BSL (cyan) display average resolutions of 2.5 Å, 2.9 Å, 2.9 Å, 3.8 Å and 2.8 Å, respectively, on the basis of the FSC value of 0.143. f, Two overall views of the EM density map. The local resolutions are color-coded for different regions of U2 snRNP. g, Angular distribution of the particles used for the reconstruction. h, The Fourier-shell correlation (FSC) curves for cross-validation between the model and the cryo-EM map of human 17S U2 snRNP.
Extended Data Fig. 2 Purification and cryo-EM reconstruction of human pre-A complex.
a, Analysis of RNA components on 8% urea-PAGE gel after glycerol gradient centrifugation. The fractions that contain pre-mRNA, U1 and U2 snRNAs are indicated by a red rectangular box. Fractions 11–15, thought to contain the pre-A complex, were pooled for cryo-EM analysis. b, Analysis of the purified pre-A complex on a silver-stained SDS-PAGE gel. The protein components were identified by mass spectrometry. c, A representative cryo-EM micrograph of the pre-A complex sample. d, Representative 2D class averages. Scale bar: 44 nm. The experiments were repeated independently for more than three times with similar results. e, A flow chart diagram of cryo-EM data processing for human pre-A complex. Please refer to Methods for details. f, The final reconstruction for the masked U2 region displays an average resolution of 3.0 Å on the basis of the FSC value of 0.143. g, Two overall views of the EM density map. The local resolutions are color-coded for different regions of U2 snRNP in the pre-A complex. h, Angular distribution of the particles used for the reconstruction of U2 snRNP region of human pre-A complex. i, The Fourier-shell correlation (FSC) curves for cross-validation between the model and the cryo-EM map for the human pre-A complex.
Extended Data Fig. 3 The EM density map of human 17S U2 snRNP.
a, The EM density map for the small molecule E7107 and its surrounding structural elements. Two related views are shown. b, The EM density map for the RRM domain of the splicing factor TAT-SF1 (left panel). The EM density maps for two representative α-helices are shown (middle and right panels). c, The EM density map for the Linker domain of TAT-SF1. d, The EM density map for SF3A2. The separator helix is indicated by a black dashed box. e, The EM density map for the α1 helix of PRP5 and its interacting elements from SF3B1. Two related views are shown. f, The EM density map for the acidic loop of PRP5 and its interacting elements from SF3B1. Two related views are shown. g, The EM density map for the α2 helix of PRP5 and its interacting elements from SF3B1. Two related views are shown.
Extended Data Fig. 4 The EM density map for different regions of the human pre-A complex.
a, The EM density map for the initial U2/BS duplex in the pre-A complex. b, The EM density map for the zinc finger (ZnF) of SF3A2. c, The EM density map for the structurally resolved region of DNAJC8. Two related views are shown. d, Docking of SF1 into the EM density map. e, Docking of U1 snRNP into the EM density map. f, Superimposition of the initial U2/BS duplex from pre-A complex and the three-way junction of U2 snRNA from human 17S U2 snRNP (grey). g, The low-pass filtered density map for the 5′-end sequences of U2 snRNA can be traced to the helicase domain of PRP5.
Extended Data Fig. 5 PRP5 knockdown in HEK293F cells induces altered splicing site selection.
a, Analysis of PRP5 mRNA level (top panel) and protein expression level (bottom panel) in control (NC) and siRNA-transfected cells (mean ± s.d., n = 3 independent replicates). b, The volcano plot of log2 fold change versus log10(Pvalue) of gene expression level in siRNA-transfected cells and WT cells. Red and blue dots indicate up-regulated and down-regulated genes, respectively (Pvalue < 0.001). c, The volcano plot of log2 fold change of ∆PSI versus log10(Pvalue) of all splicing changes. Red dots indicate significantly altered splicing events (|∆PSI| > 10 and Pvalue < 0.05). d, Distribution of novel (orange) and known (grey) aberrant splicing events in PRP5 knockdown cells. Intron retention and alternative 3’SS are the most frequently occurring events. See also Extended Data Table 4. e, Validation of representative aberrantly spliced genes in control (n = 3) and PRP5 knockdown (n = 5) cells through quantitative RT-PCR assays. f, A schematic diagram of normal and aberrant splicing of ANKHD1, a representative gene that is sensitive to PRP5 knockdown. Positions of the cryptic (red) and canonical (green) 3’SS are indicated. P values were calculated by a two-sided two-test without adjustment.
Extended Data Fig. 6 The open and closed conformations of SF3B1.
a, The open conformation of SF3B1. SF3B1 exists in an open conformation in both 17S U2 snRNP and the pre-A complex. Shown here is an overlay of the SF3B1 structures from these two complexes. b, The closed conformation of SF3B1. Pre-mRNA engagement by U2 snRNP results in a closed conformation of SF3B1. Shown here is the structure of SF3B1 from the human Bact complex. c, Structural alignment using HEAT repeats 16 through 20 of SF3B1 reveals major conformational differences between the open and closed states of SF3B1. Two perpendicular views are shown. d, Structural alignment using HEAT repeats 4 through 9 of SF3B1 reveals major conformational differences between the open and closed states of SF3B1.
Extended Data Fig. 7 Structural comparison between the yeast intron-defined pre-A complex and the human exon-defined pre-A complex.
a, Structure of the pre-A complex from S. cerevisiae. The yeast complex was assembled on an intron-definition pre-mRNA. A schematic diagram of the pre-mRNA and its recognition by the pre-A complex is shown above. b, Structure of the human pre-A complex. The human complex was assembled on an exon-definition pre-mRNA. A schematic diagram of the pre-mRNA and its recognition by the pre-A complex is shown above.
Extended Data Fig. 8 The relative orientations of U1 and U2 snRNPs in yeast and human spliceosomes.
a, Molecular organizations of yeast U1 (pink) and U2 (green and light blue) snRNPs in the pre-A, A and pre-B complexes. Overall, the U1 snRNP moves away from U2 snRNP to allow the recruitment of tri-snRNP. b, In human, the cross-exon pre-A complex is expected to be converted to cross-intron complex for splicing reaction to proceed; this step may involve interaction of U2 snRNP with another U1 snRNP that bound an upstream 5′SS.
Extended Data Fig. 9 Recognition of the splicing inhibitors E7107 and SSA by the Hinged pocket.
a, A close-up view on the EM density of E7107 in human 17S U2 snRNP. b, A close-up view on the interface between E7107 and the Hinged pocket. The Hinged pocket is exactly where the BPA binds in the assembled spliceosomes. c, A close-up view on the EM density of SSA in the human pre-A complex. SSA is covalently linked to Cys26 of PHF5A. d, A close-up view on the interface between SSA and the Hinged pocket.
Supplementary information
Supplementary Information
Supplementary Tables 1–3.
Supplementary Table 4
List of aberrant splicing events associated with PRP5 knockdown in HEK293F cells.
Source data
Source Data Fig. 6
Unprocessed western blots.
Source Data Extended Data Fig. 1
Unprocessed gels.
Source Data Extended Data Fig. 2
Unprocessed gels.
Source Data Extended Data Fig. 5
Unprocessed western blots.
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Zhang, X., Zhan, X., Bian, T. et al. Structural insights into branch site proofreading by human spliceosome. Nat Struct Mol Biol 31, 835–845 (2024). https://doi.org/10.1038/s41594-023-01188-0
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DOI: https://doi.org/10.1038/s41594-023-01188-0
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