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- PDB-9ze3: Cryo-EM structure of the endogenous U2/branchpoint spliceosomal c... -

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Basic information

Entry
Database: PDB / ID: 9ze3
TitleCryo-EM structure of the endogenous U2/branchpoint spliceosomal complex (Distal DHX15 state)
Components
  • (Splicing factor 3A subunit ...) x 3
  • (Splicing factor 3B subunit ...) x 6
  • ATP-dependent RNA helicase DHX15
  • PHD finger-like domain-containing protein 5A
  • Pre-mRNA
  • RNA-binding protein 5
  • U2 snRNA
  • U2 snRNP-associated SURP motif-containing protein
KeywordsSPLICING / spliceosome / tumor suppressor / helicase / DHX15 / RBM5 / SR140 / prespliceosomal A complex
Function / homology
Function and homology information


U11/U12 snRNP / B-WICH complex / U12-type spliceosomal complex / ATP-dependent activity, acting on RNA / response to alkaloid / RNA splicing, via transesterification reactions / blastocyst formation / splicing factor binding / U2-type precatalytic spliceosome / mRNA cis splicing, via spliceosome ...U11/U12 snRNP / B-WICH complex / U12-type spliceosomal complex / ATP-dependent activity, acting on RNA / response to alkaloid / RNA splicing, via transesterification reactions / blastocyst formation / splicing factor binding / U2-type precatalytic spliceosome / mRNA cis splicing, via spliceosome / U2-type prespliceosome assembly / U2-type spliceosomal complex / SAGA complex / U2 snRNP / U2-type prespliceosome / positive regulation of transcription by RNA polymerase III / perinuclear theca / precatalytic spliceosome / regulation of alternative mRNA splicing, via spliceosome / mRNA 3'-splice site recognition / regulation of RNA splicing / mRNA Splicing - Minor Pathway / positive regulation of transcription by RNA polymerase I / spliceosomal complex assembly / U2 snRNA binding / regulation of DNA repair / RNA processing / catalytic step 2 spliceosome / mRNA Splicing - Major Pathway / RNA splicing / antiviral innate immune response / stem cell differentiation / helicase activity / spliceosomal complex / sperm end piece / centriole / mRNA splicing, via spliceosome / positive regulation of neuron projection development / negative regulation of protein catabolic process / B-WICH complex positively regulates rRNA expression / response to toxic substance / nuclear matrix / mRNA processing / double-stranded RNA binding / sperm principal piece / sperm midpiece / defense response to virus / positive regulation of canonical NF-kappaB signal transduction / RNA helicase activity / defense response to bacterium / nuclear speck / RNA helicase / positive regulation of apoptotic process / chromatin remodeling / negative regulation of cell population proliferation / mRNA binding / apoptotic process / positive regulation of DNA-templated transcription / protein-containing complex binding / nucleolus / ATP hydrolysis activity / positive regulation of transcription by RNA polymerase II / DNA binding / RNA binding / zinc ion binding / nucleoplasm / ATP binding / nucleus / cytosol
Similarity search - Function
: / SR140, RNA recognition motif / : / RNA-binding protein 5, RNA recognition motif 1 / RNA-binding protein 5, RNA recognition motif 2 / OCRE domain / OCRE domain / DHX15, DEXH-box helicase domain / cwf21 / CID domain ...: / SR140, RNA recognition motif / : / RNA-binding protein 5, RNA recognition motif 1 / RNA-binding protein 5, RNA recognition motif 2 / OCRE domain / OCRE domain / DHX15, DEXH-box helicase domain / cwf21 / CID domain / RPR / CID domain / CID domain profile. / SF3B6, RNA recognition motif / SF3B4, RNA recognition motif 2 / Splicing factor SF3a60 binding domain / Splicing factor SF3a60 binding domain / Cactus-binding C-terminus of cactin protein / Splicing factor 3A subunit 1, ubiquitin domain / : / Replication stress response SDE2 C-terminal / G-patch domain / mRNA splicing factor Cwf21 domain / cwf21 domain / SF3A2 domain / : / Pre-mRNA-splicing factor SF3a complex subunit 2 (Prp11) / Splicing factor SF3a60 /Prp9 subunit, C-terminal / SF3A3 domain / SF3a60/Prp9 C-terminal / Pre-mRNA-splicing factor SF3A3, of SF3a complex, Prp9 / G-patch domain profile. / SF3B4, RNA recognition motif 1 / : / Splicing factor 3A subunit 1, conserved domain / Splicing factor 3A subunit 1 / Pre-mRNA splicing factor PRP21 like protein / G-patch domain / glycine rich nucleic binding domain / : / : / Domain of unknown function DUF382 / Domain of unknown function (DUF382) / SWAP/Surp / SWAP/Surp superfamily / Surp module / SURP motif repeat profile. / Suppressor-of-White-APricot splicing regulator / Splicing factor 3B, subunit 5 / Splicing factor 3B subunit 1 / Splicing factor 3B subunit 1 / Zinc-finger of C2H2 type / PSP, proline-rich / PSP / proline-rich domain in spliceosome associated proteins / Matrin/U1-C, C2H2-type zinc finger / Zinc finger matrin-type profile. / PHF5-like / PHF5-like protein / : / Splicing factor 3B subunit 5/RDS3 complex subunit 10 / Splicing factor 3B subunit 10 (SF3b10) / Splicing factor 3B subunit 1-like / ENTH/VHS / : / PPP2R1A-like HEAT repeat / SAP motif profile. / SAP domain / Putative DNA-binding (bihelical) motif predicted to be involved in chromosomal organisation / SAP domain / : / Helicase associated domain (HA2), ratchet-like / DEAD-box helicase, OB fold / Oligonucleotide/oligosaccharide-binding (OB)-fold / Helicase associated domain (HA2), winged-helix / Helicase-associated domain / Helicase associated domain (HA2) Add an annotation / Matrin/U1-C-like, C2H2-type zinc finger / U1-like zinc finger / Zinc finger domain / Zn-finger in Ran binding protein and others / Zinc finger RanBP2 type profile. / Zinc finger, RanBP2-type superfamily / Zinc finger RanBP2-type signature. / DNA/RNA helicase, ATP-dependent, DEAH-box type, conserved site / DEAH-box subfamily ATP-dependent helicases signature. / Zinc finger, RanBP2-type / : / RSE1/DDB1/CPSF1 second beta-propeller / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / : / CPSF A subunit region / RSE1/DDB1/CPSF1 first beta-propeller / Zinc finger C2H2 type domain profile. / Zinc finger C2H2 superfamily / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Zinc finger C2H2-type / RNA recognition motif
Similarity search - Domain/homology
RNA / RNA (> 10) / U2 snRNP-associated SURP motif-containing protein / ATP-dependent RNA helicase DHX15 / Splicing factor 3B subunit 1 / RNA-binding protein 5 / Splicing factor 3A subunit 3 / Splicing factor 3B subunit 2 / Splicing factor 3B subunit 3 / Splicing factor 3B subunit 4 ...RNA / RNA (> 10) / U2 snRNP-associated SURP motif-containing protein / ATP-dependent RNA helicase DHX15 / Splicing factor 3B subunit 1 / RNA-binding protein 5 / Splicing factor 3A subunit 3 / Splicing factor 3B subunit 2 / Splicing factor 3B subunit 3 / Splicing factor 3B subunit 4 / Splicing factor 3A subunit 2 / Splicing factor 3A subunit 1 / PHD finger-like domain-containing protein 5A / Splicing factor 3B subunit 5 / Splicing factor 3B subunit 6
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.93 Å
AuthorsLiu, S. / Su, T. / Zhou, Z.H.
Funding support United States, 6items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM071940 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)S10RR23057 United States
National Institutes of Health/Office of the DirectorS10OD018111 United States
National Science Foundation (NSF, United States)DBI-1338135 United States
National Science Foundation (NSF, United States)DMR-1548924 United States
National Institutes of Health/National Center for Advancing Translational Sciences (NIH/NCATS)UCLA CTSI Grant UL1TR001881 United States
CitationJournal: bioRxiv / Year: 2026
Title: The tumour suppressor RBM5 activates the helicase DHX15 to regulate splicing.
Authors: Shiheng Liu / Tiantian Su / Jeffrey Huang / Chia-Ho Lin / Douglas L Black / Andrey Damianov / Z Hong Zhou
Abstract: Pre-mRNA splicing determines the expressed proteome and is frequently dysregulated in cancer. The tumour-suppressor RBM5 controls an exon network regulating apoptosis, yet its molecular mechanism is ...Pre-mRNA splicing determines the expressed proteome and is frequently dysregulated in cancer. The tumour-suppressor RBM5 controls an exon network regulating apoptosis, yet its molecular mechanism is elusive. Using in vivo spliceosome capture and cryogenic electron microscopy, we determined structures of precatalytic spliceosomes arrested by RBM5 immediately after U2 snRNP branchpoint recognition. Despite intron diversity, the U2-pre-mRNA duplex, branchpoint adenine, and downstream polypyrimidine tract are well-resolved. RBM5 binds the outer SF3B1 HEAT surface and performs dual functions: First, its helix-loop-helix motif and upstream zinc-finger domain sterically block tri-snRNP and Prp8 docking and prevent progression to pre-B and B complexes; Second, its G-patch activates DHX15 and places this DExH-box helicase on the pre-mRNA as it exits SF3B1, poised for branch helix unwinding. DHX15 binding to SF3B1 is facilitated by U2SURP/SR140, which engages SF3B1 near RBM5's helix-loop-helix. Functional assays confirm that disruption of the RBM5 interfaces with either DHX15 or SF3B1 inhibit exon repression. Mutations at these regulatory interfaces are common in cancer genomes and predicted to disrupt its regulation of apoptotic isoforms. Thus, RBM5 acts as a dual-action spliceosome gatekeeper that couples helicase activation with physical stalling to enforce tumour-suppressive alternative splicing programmes.
History
DepositionNov 27, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 15, 2026Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RNA-binding protein 5
B: ATP-dependent RNA helicase DHX15
C: U2 snRNP-associated SURP motif-containing protein
2: U2 snRNA
H: PHD finger-like domain-containing protein 5A
R: Pre-mRNA
B1: Splicing factor 3B subunit 1
B2: Splicing factor 3B subunit 2
B3: Splicing factor 3B subunit 3
B4: Splicing factor 3B subunit 4
B5: Splicing factor 3B subunit 5
B6: Splicing factor 3B subunit 6
A1: Splicing factor 3A subunit 1
A2: Splicing factor 3A subunit 2
A3: Splicing factor 3A subunit 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)989,40318
Polymers989,20715
Non-polymers1963
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 4 types, 4 molecules ABCH

#1: Protein RNA-binding protein 5 / Protein G15 / Putative tumor suppressor LUCA15 / RNA-binding motif protein 5 / Renal carcinoma ...Protein G15 / Putative tumor suppressor LUCA15 / RNA-binding motif protein 5 / Renal carcinoma antigen NY-REN-9


Mass: 93410.133 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RBM5, H37, LUCA15 / Production host: Homo sapiens (human) / References: UniProt: P52756
#2: Protein ATP-dependent RNA helicase DHX15 / ATP-dependent RNA helicase #46 / DEAH box protein 15 / Splicing factor Prp43 / hPrp43


Mass: 91065.805 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O43143, RNA helicase
#3: Protein U2 snRNP-associated SURP motif-containing protein / 140 kDa Ser/Arg-rich domain protein / U2-associated protein SR140


Mass: 118503.312 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O15042
#5: Protein PHD finger-like domain-containing protein 5A / PHD finger-like domain protein 5A / Splicing factor 3B-associated 14 kDa protein / SF3b14b


Mass: 12427.524 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q7RTV0

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RNA chain , 2 types, 2 molecules 2R

#4: RNA chain U2 snRNA


Mass: 11784.973 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)
#6: RNA chain Pre-mRNA


Mass: 13447.404 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human)

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Splicing factor 3B subunit ... , 6 types, 6 molecules B1B2B3B4B5B6

#7: Protein Splicing factor 3B subunit 1 / Pre-mRNA-splicing factor SF3b 155 kDa subunit / SF3b155 / Spliceosome-associated protein 155 / SAP 155


Mass: 146024.938 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O75533
#8: Protein Splicing factor 3B subunit 2 / Pre-mRNA-splicing factor SF3b 145 kDa subunit / SF3b145 / Spliceosome-associated protein 145 / SAP 145


Mass: 100377.812 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q13435
#9: Protein Splicing factor 3B subunit 3 / Pre-mRNA-splicing factor SF3b 130 kDa subunit / SF3b130 / STAF130 / Spliceosome-associated protein ...Pre-mRNA-splicing factor SF3b 130 kDa subunit / SF3b130 / STAF130 / Spliceosome-associated protein 130 / SAP 130


Mass: 135718.844 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q15393
#10: Protein Splicing factor 3B subunit 4 / Pre-mRNA-splicing factor SF3b 49 kDa subunit / Spliceosome-associated protein 49 / SAP 49


Mass: 44436.570 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q15427
#11: Protein Splicing factor 3B subunit 5 / SF3b5 / Pre-mRNA-splicing factor SF3b 10 kDa subunit


Mass: 10149.369 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9BWJ5
#12: Protein Splicing factor 3B subunit 6 / Pre-mRNA branch site protein p14 / SF3b 14 kDa subunit / SF3B14a / Spliceosome-associated protein / ...Pre-mRNA branch site protein p14 / SF3b 14 kDa subunit / SF3B14a / Spliceosome-associated protein / 14-kDa / Splicing factor 3b / subunit 6 / 14kDa


Mass: 14606.900 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9Y3B4

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Splicing factor 3A subunit ... , 3 types, 3 molecules A1A2A3

#13: Protein Splicing factor 3A subunit 1 / SF3a120 / Spliceosome-associated protein 114 / SAP 114


Mass: 88991.094 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q15459
#14: Protein Splicing factor 3A subunit 2 / SF3a66 / Spliceosome-associated protein 62 / SAP 62


Mass: 49327.355 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q15428
#15: Protein Splicing factor 3A subunit 3 / SF3a60 / Spliceosome-associated protein 61 / SAP 61


Mass: 58934.844 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q12874

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Non-polymers , 1 types, 3 molecules

#16: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Endogenous U2/branchpoint spliceosomal complex (Distal DHX15)
Type: COMPLEX / Entity ID: #1-#15 / Source: NATURAL
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.9
Details: 20 mM HEPES-KOH pH 7.9, 150 mM NaCl, 1.5 mM MgCl2, 10 mM DTT, 4.5% glycerol
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1RELIONparticle selection
2cryoSPARCparticle selection
3PHENIX1.20.1_4487model refinement
14cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 49971 / Symmetry type: POINT
Atomic model buildingB value: 119.5 / Space: REAL

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