The U2 small nuclear ribonucleoprotein (snRNP) has an essential role in the selection of the precursor mRNA branch-site adenosine, the nucleophile for the first step of splicing1. Stable addition of U2 during early spliceosome formation requires the DEAD-box ATPase PRP52,3,4,5,6,7. Yeast U2 small nuclear RNA (snRNA) nucleotides that form base pairs with the branch site are initially sequestered in a branchpoint-interacting stem–loop (BSL)8, but whether the human U2 snRNA folds in a similar manner is unknown. The U2 SF3B1 protein, a common mutational target in haematopoietic cancers9, contains a HEAT domain (SF3B1HEAT) with an open conformation in isolated SF3b10, but a closed conformation in spliceosomes11, which is required for stable interaction between U2 and the branch site. Here we report a 3D cryo-electron microscopy structure of the human 17S U2 snRNP at a core resolution of 4.1 Å and combine it with protein crosslinking data to determine the molecular architecture of this snRNP. Our structure reveals that SF3B1HEAT interacts with PRP5 and TAT-SF1, and maintains its open conformation in U2 snRNP, and that U2 snRNA forms a BSL that is sandwiched between PRP5, TAT-SF1 and SF3B1HEAT. Thus, substantial remodelling of the BSL and displacement of BSL-interacting proteins must occur to allow formation of the U2–branch-site helix. Our studies provide a structural explanation of why TAT-SF1 must be displaced before the stable addition of U2 to the spliceosome, and identify RNP rearrangements facilitated by PRP5 that are required for stable interaction between U2 and the branch site.
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The atomic coordinate files have been deposited in the Protein Data Bank (PDB) with the following accession codes: U2 5′ domain (6Y50), low resolution region (6Y53) and entire 17S U2 particle (6Y5Q). The cryo-EM maps have been deposited in the Electron Microscopy Data Bank as follows: U2 5′ domain (EMD-10688) and entire U2 particle (EMD-10689).
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We thank T. Conrad for the production of HeLa cells in a bioreactor, H. Kohansal for preparing HeLa cell nuclear extract, and G. Heyne, U. Steuerwald, W. Lendeckel, M. Raabe and U. Pleßmann for technical assistance. This work was supported by the Max Planck Society and the Deutsche Forschungsgemeinschaft (SFB 860 grants to H.U., R.L. and H.S.).
The authors declare no competing interests.
Peer review information Nature thanks Aaron Hoskins, Patrick Schultz, Jonathan Staley and the other, anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Spliceosome assembly cycle and the interaction of 17S U2 with the pre-mRNA branch site.
a, Early assembly and catalytic activation pathway of the spliceosome. The 17S U2 snRNP, the structure of which was determined here by cryo-EM, is indicated by an asterisk. For simplicity, the stepwise interactions of the U1, U2, U4/U6 and U5 snRNPs, and only selected non-snRNP proteins are shown. Helicases involved in conversion of the E to A complex are indicated. In the E complex, the U2AF65 UHM interacts with the ULM of SF1, and after release of SF1, it subsequently interacts with a ULM in the N-terminal region of SF3B1. This swap of UHM–ULM interactions is probably very important for positioning the BS before the conformational change in the HEAT domain clamps down on the U2–BS helix and stabilizes the U2 snRNP interaction with the intron. The U2AF65/U2AF35 dimer is released (not shown) during conversion of the A to B complex20. SF1 is displaced from the BS by UAP56 (either before or after PRP5 action). SF1 pre-bulges the BS-A via accommodation of the latter in its KH domain, facilitating subsequent base-pairing of U2 with the BS29. b, Base-pairing interactions between U2 snRNA and the BS and upstream intron nucleotides, that lead to bulging of the BS-A. The sequence shown is from intron 10 of the pre-mRNA for the polypyrimidine tract-binding protein (PTB). Red shading denotes the bona fide U2–BS helix, and shows base pairs formed between human U2 snRNA and the conserved BS consensus sequence of PTB intron 10. Yellow shading denotes the extended U2–BS helix, in which the number and nature of base-pairing interactions varies depending on the pre-mRNA intron sequence. c, Schematic of the composition of the human 17S U2 snRNP. Only abundant U2 proteins are shown20. d, Open structure of the SF3B1 HEAT domain in the isolated SF3b complex (left) and its more closed conformation after interaction with the U2–BS helix (right). The SF3B1 HEAT domain (green) forms a super-helical structure, and in the spliceosome sequesters the U2–BS helix. The conformational change in the HEAT domain, which is required to form the BS-A binding pocket, was proposed to occur after formation of the U2–BS helix17. Before this study, the conformation of SF3B1 in human 17S U2 snRNP was not known. The pre-mRNA is in grey; U2 snRNA is coloured as in Fig. 2. For simplicity, the PHF5A protein that also forms part of the BS-A binding pocket is not shown. The structures of SF3B1HEAT in the isolated SF3b complex (PDB code 5IFE) (left) and human Bact complex (PDB code 6FF4) (right) are aligned via HEAT repeat 20.
Extended Data Fig. 2 Cryo-EM and image-processing of the human 17S U2 snRNP.
a, Computation sorting scheme. All major image-processing steps are depicted. For a more detailed explanation, see ‘Image processing’ in the Methods. A considerable amount of conformational heterogeneity is present in all spliceosomal complexes but even more in the bipartite 17S U2 snRNP, which is structurally very labile and readily dissociates during purification43, making its analysis by electron microscopy challenging. In addition, the bridges connecting the 5′ and 3′ domains of the U2 particle (see also Extended Data Fig. 5) have a very flexible character, leading to flexibility in the 3′ domain and the large variation in local resolution. Thus, a substantially higher number of particles was needed to generate the 17S U2 structure than is usually used for cryo-electron microscopy. b, Typical cryo-electron micrograph of the Homo sapiens 17S U2 snRNP recorded at 120,700× magnification with a Titan Krios microscope using a Falcon III direct electron detector operating in integration mode at a calibrated pixel size of 1.16 Å. c, Representative selection of reference-free 2D class average images depicting 17S U2 particles recorded under cryo conditions. d, Euler angle distribution plot of all 17S U2 particles that contributed to the final structure. Red depicts a higher relative number of particles at a certain angle. The generally uniform distribution of the particle projection angles ensures an isotropic 3D electron-microscopy density map. e, Local resolution estimation of the 5′ domain of 17S U2 snRNP. The 5′ domain shows a resolution between 3.6 and 9.0 Å. The map of the remaining part excluding the 5′ domain, shown as a translucent overlay, was determined at resolutions between 10 and 30 Å. f, Fourier shell correlation (FSC) of two independently refined half datasets, calculated using the ‘PostProcessing’ routine in RELION, indicates a global resolution of 7.1 Å for the entire 17S U2 snRNP, and 4.1 Å for the masked U2 5′ domain. Multibody refinement around the 3′ domain and the peripheral parts did not produce better resolved maps for those regions. g, Map versus model FSC curves for the 5′ domain and the SF3b core. The FSC = 0.5 criterion indicates a resolution of 4.2 Å for the U2 5′ domain, and 4.15 Å for the SF3b core.
Extended Data Fig. 3 Euclidian distances for crosslinks observed between modelled residues (Cα) of the 17S U2 snRNP.
a–c, Crosslinks from a single 17S U2 crosslinking experiment (with two technical replicates) were identified by pLink2.3.5 and filtered to a false discovery rate (FDR) of 1% (a), pLink1.23 at an FDR of 1% (b), and pLink1.23 at an FDR of 5% (c). Calculations were performed using PyMOL2.3.4 for crosslinks with a score of at least 1. Most crosslinks (93–95% at the spectral level and 85–86% at the unique crosslink level) are consistent—that is, Cα atoms of the crosslinked amino acids are within 30 Å of each other—in the presented model of the 17S U2 snRNP. The percentage of overlength crosslinks (that is, longer than 30 Å) is slightly higher than observed for more rigid complexes, which is consistent with the known structural flexibility/dynamics of the 17S U2 snRNP.
Extended Data Fig. 4 The SF3B1 HEAT domain has an open conformation in the 17S U2 snRNP.
a, Fit of the SF3b core proteins into the 17S U2 electron-microscopy density (grey). b, Overlay of the HEAT domain (amino acids 529–1201) of SF3B1 in 17S U2 snRNP (green) and the crystal structure of the isolated SF3b core (gold, PDB 5IFE). In 17S U2 and isolated SF3b, PHF5A contacts both N- and C-terminal regions of the HEAT domain, interacting with HEAT repeats HR2–HR3 near its N terminus, as well as HR15, HR17 and HR18 near its C terminus. PHF5A thus contacts two previously described, dynamic hinge regions (HR3–HR4 and HR15–HR16) of the HEAT domain10, and thereby helps to stabilize the SF3B1 open conformation. c, Close up of SF3B1 HR16 in 17S U2 overlaid with that in isolated SF3b. HR16 is completely structured in the 17S U2 particle, but not in isolated SF3b. d, Overlay of the SF3b core domain in 17S U2 snRNP (green) with the crystal structure of isolated SF3b (gold; PDB code 5IFE). For clarity, the SF3B3 protein is coloured red-orange in this panel. Although the WD40-A and WD40-C domains of SF3B3 have essentially the same conformation, and clamp SF3B5 in a similar manner in both the 17S U2 snRNP and the SF3B core complex, the WD40-B domain has a slightly different position and is rotated more towards SF3B1HEAT in 17S U2. e, Multiple crosslinks were detected between SF3B6, which can be crosslinked to the BS-A in spliceosomal A complexes44,45, and SF3B1 residues on the upper surface of the HEAT domain, as well as the PRP5 α-helix that interacts with HR9–HR12, and PHF5A (see also Supplementary Table 1). Crosslinks between SF3B1 and SF3B6 were also detected in the N-terminal region of SF3B1 located at, or near, amino acids (373–415) that are required for stable SF3B6–SF3B1 interaction45,46. Similar protein–protein crosslinks involving SF3B6 were observed with recombinant, intact SF3b complexes10, which indicates that SF3B6 is located in a similar, but not firmly fixed, position both in 17S U2 and the isolated SF3b complex10. Numbers (colour-coded to match protein colours) indicate the positions of crosslinked lysine residues, which are connected by black arrows. The distances between the crosslinked residues in our 17S U2 model are indicated by small numbers next to the black arrows. A distance is not included if one of the crosslinked residues is present in an unstructured protein region. f, The site where the splicing modulator pladienolide B (PlaB) binds SF3B1 is not occupied in the 17S U2 snRNP. The crystal structure of PlaB bound to the SF3b core complex showed that the binding pocket of PlaB, which is formed by HR15–HR17 and PHF5A, is present only in the open conformation of the HEAT domain, and overlaps with the BS-A-binding pocket17. As the PlaB-binding pocket is present in a hinge region of the HEAT domain, it was proposed to inhibit SF3b function by preventing the conformational change in the HEAT domain needed to clamp down on the U2–BS helix17. There is no electron-microscopy density observed in the PlaB-binding pocket and thus it and potentially other splicing modulators can also bind SF3B1 in the 17S U2 snRNP.
Extended Data Fig. 5 The 3′ domain of the 17S U2 snRNP and molecular bridges connecting it to SF3b.
a, The U2 3′ domain is connected to the 5′ domain by three main bridges. Fit of the entire 17S U2 molecular model into the electron-microscopy density (low-pass filtered). b, Fit of the U2 Sm core, U2 snRNA SLIII, and U2-A' and U2-B” bound to U2 snRNA SLIV. The overall structure of the U2 3′ domain does not change substantially after U2 incorporation into the spliceosome. The U2 Sm core domain is located at a similar distance from SF3B1HEAT as observed in the human B complex13,15. The resolution of the 17S U2 present in the yeast A complex47, as well as in the human pre-B complex15,48, is not sufficient to make meaningful, detailed comparisons of their structure with that of our 17S U2 snRNP. Furthermore, in the former complexes the molecular architecture of U2 is derived entirely from that found in yeast B or human Bact complexes. c, Bridge 1 is probably composed of U2 snRNA nucleotides upstream of the Sm-binding site that connect it to SLIIb, which is also part of this bridge, as well as unassigned protein density. d, Bridge 2 is formed by RRM2 of U2-B′′ and amino acids in the C-terminal half of SF3A3 that bridge U2-B′′ and the WD40-C domain of SF3B3. e, The N-terminal helical domain of SF3A3 contacts the U2 Sm core. SF3A3 also interacts with SF3B2 and then extends to the U2 snRNA SLIIa and BSL (see Fig. 2). Rigid-body fitting combined with protein–protein crosslinking (see f and g) allowed us to localize near or within bridge 3, both RRM1 (amino acids 13–91) and RRM2 (amino acids 100–179) of the SF3B4 protein. SF3B4RRM1 interacts with a short region of its binding partner SF3B2 (amino acids 607–693) and SF3B4RRM2 extends towards the β-sandwich of SF3A2 (amino acids 118–209) and a long helical region of SF3A1 (amino acids 235–274) that also comprises part of bridge 3. SF3A1 extends from the N-terminal region of SF3A3 to the SF3A2 β-sandwich. Thus, SF3a proteins have important bridging roles in connecting the 3′ and 5′ domains of the U2 snRNP. Stable integration of SF3a into the human 17S U2 particle during its biogenesis requires the prior binding of SF3b12 and thus the SF3a–SF3b protein contacts described above potentially have key roles in the assembly of the 17S U2 particle. f, g, Intermolecular crosslinks supporting the location in our 17S U2 model of SF3B4RRM2 (f) and SF3B4RRM1 (g). Numbers (colour-coded to match protein colours) indicate the positions of crosslinked lysine residues, which are connected by black arrows. The distance between the crosslinked residues in our 17S U2 model is indicated by small numbers next to the black arrows. A distance is not included if one of the crosslinked residues is present in an unstructured protein region.
Extended Data Fig. 6 Fit of the various stem–loops in the 5′ half of U2 snRNA.
a, b, Alternative conformations of stem–loops potentially formed in the 5′ part of human U2 snRNA. The stem of the BSL could potentially form additional base pairs, but the presence of the SF3A3 separator helix (amino acids Y392 to H400) prevents base-pair formation beyond G25–C45. This guarantees that U46 and U47 are single-stranded and thus could potentially be involved in the proposed movement of the BSL away from SF3B1 during U2–BS helix formation (see also Extended Data Fig. 8). c, Fit of U2 SLIIa, BSL and SLI three-way junction into 17S U2 electron-microscopy density. d–f, Fit of the individual SLIIa (d), BSL (e) and a shortened SLI (f) into the electron-microscopy density. Owing to the formation of the BSL, only a shortened U2 SLI can form as nucleotides in the lower stem of an extended SLI would instead form base pairs located in the lower stem of the BSL. Thus, an extended SLI and a BSL are mutually exclusive, competing U2 snRNA conformations. The position of the remaining 10 nucleotides at the 5′ end of the U2 snRNA, which in the spliceosome form part of U2–U6 helix II, cannot be discerned. g, Fit of the SF3A3 separator helix into density at the base of the BSL. h, Similar SLIIa RNP architecture in 17S U2 snRNP, and the yeast B and human Bact spliceosomal complexes. In human U2 snRNP, loop nucleotides of SLIIa contact amino acids of the loop connecting the two α-helices of SF3B1 HR20, as well as residues of SF3B2; two regions of the latter (amino acids 458–530 and 565–598) are located in well-resolved density close to SLIIa and SLI, respectively. These SLIIa contacts are similar to those found in yeast B and human Bact spliceosomes. Thus, they are a major, direct anchor point for SF3b on the U2 snRNA. The poor resolution of the cryo-EM structure of the human B complex in this region does not allow for an accurate comparison.
Extended Data Fig. 7 Fit of PRP5 and TAT-SF1 and their interaction with the SF3B1 HEAT domain.
a, Schematic of the domain organization of human PRP5 (left) and TAT-SF1 (right), with amino acid boundaries of each domain indicated below. b, Fit of the PRP5 RecA1 and RecA2 domains in an open conformation into the 17S U2 electron-microscopy density. The open (inactive) conformation of the PRP5 RecA domains seems to fit better than the closed (active) conformation, which suggests that PRP5 is inactive in 17S U2 snRNP. However, neither the resolution in this region nor intramolecular PRP5 crosslinks allow us to confidently position these two domains relative to each other and thereby distinguish between these two conformations. DEAD-box proteins typically bind double-stranded RNA via their RecA domains and facilitate local strand separation by introducing one or two sharp bends in one of the bound strands that prevent base-pairing with the complementary strand28. The resolution of the 17S U2 in the region where the PRP5 RecA domains are located does not allow us to discern whether PRP5 is interacting with RNA at this stage. On the basis of our structure, the RecA domains of PRP5 in 17S U2 are not close enough to the BSL to directly disrupt it, and also are not located close enough to the SLI. c, Protein crosslinks support the positioning of the PRP5 RecA domains and the TAT-SF1UHM. Crosslinks are observed between TAT-SF1UHM and residues in the N- and C-terminal HEAT repeats of SF3B1, in the region of PRP5 between the N-terminal α-helix and RecA domains, and in PHF5A, which supports the location of TAT-SF1UHM in the less-well resolved density element adjacent to the U2 BSL. d, Fit of the PRP5 α-helix (amino acids 146–196) into the electron-microscopy density contacting HEAT repeats 9–12. e, Protein crosslinks between PRP5 and SF3B1 suggest that a region spanning approximately 200 amino acids located N-terminal of the PRP5 helicase domain, wraps around most of SF3B1HEAT. These data are consistent with studies showing that yeast Prp5 also interacts with HR1–HR6 and HR9–HR12 of the yeast SF3B1 homologue, Hsh15523. Numbers (colour-coded to match protein colours) indicate the positions of crosslinked lysine residues, which are connected by black arrows. The proposed path of unstructured regions of PRP5 is indicated by a dotted line and the location of the conserved PRP5 DPLD motif is shown. The position of selected cancer-related hotspot mutations of SF3B1 (K666, K662, K700 and G742) are indicated. Point mutations in SF3B1HEAT are linked to various cancers and lead to the utilization of cryptic branch sites and 3′ splice sites in vivo49,50. The exact mechanism responsible for these changes in alternative splicing is currently unknown but it has been suggested that these mutations may affect the curvature of the HEAT domain10. Studies in yeast showed that position-equivalent, point mutations in SF3B1 that are linked to cancer in humans, lead to loss of stable Prp5 binding to the U2 snRNP23,25. More recent studies indicate that in humans these cancer-related mutations destabilize the interaction of SF3B1 with the SUGP1 protein51, which, however, is essentially absent from our 17S U2 preparations20. As PRP5 appears to encompass the entire HEAT domain in the human 17S U2, it is conceivable that a change in the curvature of the HEAT domain could destabilize the SF3B1–PRP5 interaction, and the absence of PRP5 may directly lead to alterations in BS selection by U2 containing SF3B1 cancer-related point mutations. Prp5 was shown to contact the SF3b complex in the U2 snRNP via a conserved DPLD motif in its N-terminal domain24. The most common (hotspot) cancer-related point mutations mainly cluster in or near HR610 and notably our crosslinking data place the DPLD motif of PRP5 (amino acids 226–229) in proximity to HR6. Thus cancer-related hotspot mutations may disrupt this essential interaction, leading to destabilization of PRP5. This, in turn, could have a detrimental effect on the function of PRP5 to facilitate the formation of a stable U2–BS interaction and/or on the proofreading activity of PRP5 (as discussed above), and thus facilitate the usage of aberrant BS and 3′ splice sites. f, Protein crosslinks between TAT-SF1RRM1 and neighbouring proteins. In 17S U2, TAT-SF1RRM1 is located adjacent to SF3B1 HR15 and HR16 (Fig. 3a), and thus may also stabilize HR16, which is completely structured in 17S U2 (Extended Data Fig. 4c). Crosslinking also suggests that an unstructured loop of SF3B2 consisting of amino acids 531–564 may occupy the electron-microscopy density near SF3B1 HR12–HR15. g, Position of SF3B1 and SF3B2 in 17S U2 (left) and in the human Bact complex (right). In 17S U2, TAT-SF1RRM1 is located in the same position where two α-helices of SF3B2 are found in the Bact complex, and thus release of TAT-SF1 would be required for the subsequent formation or repositioning of this region of SF3B2. h, Fit of TAT-SF1 UHM (amino acids 260–353) into the 17S U2 electron-microscopy density. Amino acids 260–353 of human TAT-SF1 were initially designated RRM2, but were later shown to comprise a UHM52.
Extended Data Fig. 8 RNP rearrangements and movements of the U2 snRNA required for formation of the extended U2–BS helix.
a, Spatial orientation of the BSL and neighbouring proteins in the 17S U2 (left), and the subsequently rearranged U2 snRNA and U2 proteins after formation of the U2/BS helix and stable U2 incorporation into the spliceosome (right). Although the human Bact structure (PDB code 6FF4) is shown, the spatial organization of the shown U2 components is similar in B and presumably also human A complexes. b, Movement of U2 SLI behind the BSL requires repositioning of the latter. Coloured surfaces are derived from the fitted protein models, with the RNA depicted as a combination of a transparent surface model and RNA helix. The movement of the 5′ end of U2 required for formation of the extended U2–BS helix would require release of the BSL from SF3B1. In the absence of the latter, the 5′ end of U2 snRNA, including the short SLI, which is topologically located above the BSL stem, would have to be threaded through a very narrow opening between the BSL and the SF3B1 HEAT domain in order to unwind the BSL, a scenario that is unlikely. Thus, the remodelling of the U2 BSL into an extended U2–BS helix will probably occur in a conformational state of the U2 snRNP in which the BSL is moved away from the C-terminal HEAT repeats. This movement could involve a rotation around U2 nucleotides U46 and/or U47, which link the BSL to the stem of SLIIa and appear to be maintained in a single-stranded conformation by SF3A3 (Extended Data Fig. 6). In this respect, it is intriguing that the short SF3A3 separator helix is situated at the same place in 17S U2 and in human Bact complexes. Moreover, it probably has very similar roles in 17S U2 and the spliceosome. That is, in Bact, the SF3A3 separator helix also determines the length of the extended part of the U2–BS helix and facilitates the movement of U2 snRNA and the intron away from each other. The SF3A3 separator helix probably maintains its contact with the U2 SLIIa, ensuring the stable interaction of the SF3b complex with the U2 snRNA. This has the advantage that the newly formed extended U2–BS complex can swing back towards the HEAT domain with SF3A3 still bound to the SLIIa. Docking of the extended U2–BS helix to the SF3B1 C-terminal HEAT repeats is potentially facilitated by initial interactions with the backbone of the first 5–6 nucleotides of the intron downstream of the BS. c, Movements of the 5′ end of U2 that enable formation of the extended U2–BS helix. Step 1: ATP hydrolysis by PRP5 disrupts protein contacts with the BSL and presumably also with the 5′ end of the U2 snRNA including SLI. This allows base pairing between the BSL loop nucleotides and the BS of the pre-mRNA intron. Step 2: the destabilized BSL unwinds and the 5′ end of U2 moves behind the BSL and downward, with a twisting motion that repositions the pre-mRNA behind the unwound BSL. Movement of the 5′ end downward allows the formation of additional base pairs with the pre-mRNA. As both ends of the pre-mRNA intron appear to be fixed by protein and snRNP interactions, formation of the helical conformation of the U2–BS and extended U2–BS probably involves first movement of the 5′ end of U2 behind the pre-mRNA (step 4), followed by a movement across the pre-mRNA (that is, a twisting rotation of the 5′ end of U2 around the pre-mRNA) (step 5). Numbers indicate the position of selected U2 nucleotides.
Supplementary Table 1
Protein-protein crosslinks identified in human 17S U2 snRNPs. Crosslinks identified by pLink1.23 and pLink2.3.5 with a score of at least 1 are shown. The number of crosslink-spectrum matches (CSMs) and the highest score are indicated for each crosslinked peptide. "Residue 1" and "Residue 2" are the crosslinked residue pairs in the Protein 1 and Protein 2, respectively.
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Zhang, Z., Will, C.L., Bertram, K. et al. Molecular architecture of the human 17S U2 snRNP. Nature 583, 310–313 (2020). https://doi.org/10.1038/s41586-020-2344-3
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