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- PDB-9oxs: CRYO-EM STRUCTURE OF the human mPSF IN COMPLEX WITH THE AAUAAU po... -

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Basic information

Entry
Database: PDB / ID: 9oxs
TitleCRYO-EM STRUCTURE OF the human mPSF IN COMPLEX WITH THE AAUAAU poly(A) signal
Components
  • Cleavage and polyadenylation specificity factor subunit 1
  • Isoform 2 of Cleavage and polyadenylation specificity factor subunit 4
  • RNA (5'-R(P*AP*AP*UP*AP*AP*UP*C)-3')
  • pre-mRNA 3' end processing protein WDR33
KeywordsRNA BINDING PROTEIN/RNA / polyadenylation signal / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


co-transcriptional RNA 3'-end processing, cleavage and polyadenylation pathway / Inhibition of Host mRNA Processing and RNA Silencing / Processing of Intronless Pre-mRNAs / mRNA cleavage and polyadenylation specificity factor complex / collagen trimer / mRNA 3'-UTR AU-rich region binding / mRNA 3'-end processing / Transport of Mature mRNA Derived from an Intronless Transcript / mRNA 3'-end processing / tRNA processing in the nucleus ...co-transcriptional RNA 3'-end processing, cleavage and polyadenylation pathway / Inhibition of Host mRNA Processing and RNA Silencing / Processing of Intronless Pre-mRNAs / mRNA cleavage and polyadenylation specificity factor complex / collagen trimer / mRNA 3'-UTR AU-rich region binding / mRNA 3'-end processing / Transport of Mature mRNA Derived from an Intronless Transcript / mRNA 3'-end processing / tRNA processing in the nucleus / DNA damage tolerance / RNA Polymerase II Transcription Termination / Processing of Capped Intron-Containing Pre-mRNA / fibrillar center / mRNA processing / sequence-specific double-stranded DNA binding / spermatogenesis / intracellular membrane-bounded organelle / enzyme binding / RNA binding / zinc ion binding / nucleoplasm / nucleus
Similarity search - Function
Zinc-finger CCCH domain / Zinc-finger containing family / CPSF complex subunit CPSF4-like / Pre-mRNA 3' end processing protein Pfs2-like / Zinc finger C-x8-C-x5-C-x3-H type (and similar) / Zinc finger, CCCH-type superfamily / zinc finger / Collagen triple helix repeat / Collagen triple helix repeat (20 copies) / Zinc finger, CCCH-type ...Zinc-finger CCCH domain / Zinc-finger containing family / CPSF complex subunit CPSF4-like / Pre-mRNA 3' end processing protein Pfs2-like / Zinc finger C-x8-C-x5-C-x3-H type (and similar) / Zinc finger, CCCH-type superfamily / zinc finger / Collagen triple helix repeat / Collagen triple helix repeat (20 copies) / Zinc finger, CCCH-type / Zinc finger C3H1-type profile. / 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 / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / WD domain, G-beta repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / Cleavage and polyadenylation specificity factor subunit 4 / Cleavage and polyadenylation specificity factor subunit 1 / pre-mRNA 3' end processing protein WDR33
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.07 Å
AuthorsHuang, L. / Tong, L. / Chu, H.-F.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118093 United States
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Molecular basis for the recognition of low-frequency polyadenylation signals by mPSF.
Authors: Lin Huang / Hsu-Feng Chu / Liang Tong /
Abstract: The 3'-end cleavage and polyadenylation of pre-mRNAs is dependent on a key hexanucleotide motif known as the polyadenylation signal (PAS). The PAS hexamer is recognized by the mammalian ...The 3'-end cleavage and polyadenylation of pre-mRNAs is dependent on a key hexanucleotide motif known as the polyadenylation signal (PAS). The PAS hexamer is recognized by the mammalian polyadenylation specificity factor (mPSF). AAUAAA is the most frequent PAS hexamer and together with AUUAAA, the second most frequent hexamer, account for ∼75% of the poly(A) signals. The remaining hexamers are at low frequency (<3%), and the molecular basis for their recognition is still not known. Here, we have determined the binding affinities for most of the PAS hexamers, showing that the Kd values are generally inversely correlated with their frequency. We also observed good cleavage activity for two low-frequency hexamers, AAGAAA and AACAAA. We have determined the cryo-electron microscopy structures of human mPSF in complex with AAUAAU and AGUAAA, at 3.1 and 2.5 Å resolution, respectively. The overall binding modes of the two low-frequency hexamers are similar to that of AAUAAA, although the U3-A6 Hoogsteen base pair is disrupted in the AAUAAU hexamer. For AGUAAA, the G2 base undergoes a large conformational change, which allows it to maintain the hydrogen-bonding interaction with CPSF30 as observed with A2 and establish a new hydrogen bond to CPSF30.
History
DepositionJun 4, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 26, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Cleavage and polyadenylation specificity factor subunit 1
B: pre-mRNA 3' end processing protein WDR33
C: Isoform 2 of Cleavage and polyadenylation specificity factor subunit 4
E: RNA (5'-R(P*AP*AP*UP*AP*AP*UP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)258,7177
Polymers258,5214
Non-polymers1963
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Cleavage and polyadenylation specificity factor subunit 1 / Cleavage and polyadenylation specificity factor 160 kDa subunit / CPSF 160 kDa subunit


Mass: 161074.234 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CPSF1, CPSF160 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q10570
#2: Protein pre-mRNA 3' end processing protein WDR33 / WD repeat-containing protein 33 / WD repeat-containing protein of 146 kDa


Mass: 66053.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: WDR33, WDC146 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9C0J8
#3: Protein Isoform 2 of Cleavage and polyadenylation specificity factor subunit 4 / Cleavage and polyadenylation specificity factor 30 kDa subunit / CPSF 30 kDa subunit / NS1 effector ...Cleavage and polyadenylation specificity factor 30 kDa subunit / CPSF 30 kDa subunit / NS1 effector domain-binding protein 1 / Neb-1 / No arches homolog


Mass: 27589.004 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CPSF4, CPSF30, NAR, NEB1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O95639
#4: RNA chain RNA (5'-R(P*AP*AP*UP*AP*AP*UP*C)-3')


Mass: 3804.344 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
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: human mPSF in complex with the AAUAAU poly(A) signal / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: OTHER
Electron lensMode: OTHER / Nominal magnification: 81000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 51.15 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
7Coot0.9.8.8 ELmodel fitting
13PHENIX1.21rc1_5049model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 292202 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingPDB-ID: 9OXE

9oxe


Accession code: 9OXE / Source name: PDB / Type: experimental model
RefinementHighest resolution: 3.07 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00313586
ELECTRON MICROSCOPYf_angle_d0.53918444
ELECTRON MICROSCOPYf_dihedral_angle_d5.4341851
ELECTRON MICROSCOPYf_chiral_restr0.0442054
ELECTRON MICROSCOPYf_plane_restr0.0042326

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