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- PDB-8e3i: CRYO-EM STRUCTURE OF the human MPSF IN COMPLEX WITH THE AUUAAA po... -

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Basic information

Entry
Database: PDB / ID: 8e3i
TitleCRYO-EM STRUCTURE OF the human MPSF IN COMPLEX WITH THE AUUAAA poly(A) signal
Components
  • Cleavage and polyadenylation specificity factor subunit 1
  • Cleavage and polyadenylation specificity factor subunit 4
  • RNA (5'-R(P*CP*AP*UP*UP*AP*AP*AP*CP*AP*AP*C)-3')
  • pre-mRNA 3' end processing protein WDR33
KeywordsRNA BINDING PROTEIN / mRNA / 3'processing / polyadenylation / CPSF
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 / mRNA 3'-UTR AU-rich region binding / collagen trimer / mRNA 3'-end processing / Transport of Mature mRNA Derived from an Intronless Transcript / postreplication repair / 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 / mRNA 3'-UTR AU-rich region binding / collagen trimer / mRNA 3'-end processing / Transport of Mature mRNA Derived from an Intronless Transcript / postreplication repair / tRNA processing in the nucleus / 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 / Zinc finger, CCCH-type / Zinc finger C3H1-type profile. / Collagen triple helix repeat ...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 / Zinc finger, CCCH-type / Zinc finger C3H1-type profile. / Collagen triple helix repeat / Collagen triple helix repeat (20 copies) / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / Mono-functional DNA-alkylating methyl methanesulfonate N-term / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / CPSF A subunit region / zinc finger / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / 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: 2.53 Å
AuthorsGutierrez, P.A. / Wei, J. / Sun, Y. / Tong, L.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118093 United States
CitationJournal: RNA / Year: 2022
Title: Molecular basis for the recognition of the AUUAAA polyadenylation signal by mPSF.
Authors: Pedro A Gutierrez / Jia Wei / Yadong Sun / Liang Tong /
Abstract: The polyadenylation signal (PAS) is a key sequence element for 3'-end cleavage and polyadenylation of messenger RNA precursors (pre-mRNAs). This hexanucleotide motif is recognized by the mammalian ...The polyadenylation signal (PAS) is a key sequence element for 3'-end cleavage and polyadenylation of messenger RNA precursors (pre-mRNAs). This hexanucleotide motif is recognized by the mammalian polyadenylation specificity factor (mPSF), consisting of CPSF160, WDR33, CPSF30, and Fip1 subunits. Recent studies have revealed how the AAUAAA PAS, the most frequently observed PAS, is recognized by mPSF. We report here the structure of human mPSF in complex with the AUUAAA PAS, the second most frequently identified PAS. Conformational differences are observed for the A1 and U2 nucleotides in AUUAAA compared to the A1 and A2 nucleotides in AAUAAA, while the binding modes of the remaining 4 nt are essentially identical. The 5' phosphate of U2 moves by 2.6 Å and the U2 base is placed near the six-membered ring of A2 in AAUAAA, where it makes two hydrogen bonds with zinc finger 2 (ZF2) of CPSF30, which undergoes conformational changes as well. We also attempted to determine the binding modes of two rare PAS hexamers, AAGAAA and GAUAAA, but did not observe the RNA in the cryo-electron microscopy density. The residues in CPSF30 (ZF2 and ZF3) and WDR33 that recognize PAS are disordered in these two structures.
History
DepositionAug 17, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 18, 2023Provider: 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
C: Cleavage and polyadenylation specificity factor subunit 4
E: RNA (5'-R(P*CP*AP*UP*UP*AP*AP*AP*CP*AP*AP*C)-3')
B: pre-mRNA 3' end processing protein WDR33
hetero molecules


Theoretical massNumber of molelcules
Total (without water)258,2877
Polymers258,0904
Non-polymers1963
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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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 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: 27646.055 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: Escherichia coli (E. coli) / References: UniProt: O95639
#3: RNA chain RNA (5'-R(P*CP*AP*UP*UP*AP*AP*AP*CP*AP*AP*C)-3')


Mass: 3458.154 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#4: Protein pre-mRNA 3' end processing protein WDR33 / WD repeat-containing protein 33 / WD repeat-containing protein of 146 kDa


Mass: 65912.039 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
#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

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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-AUUAAA RNA complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8 / Details: 25 mM Tris (pH 8.0), 150 mM NaCl, and 5 mM DTT
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: -2000 nm / Nominal defocus min: -1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 58 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4607

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4332000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.53 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 858038 / Symmetry type: POINT
Atomic model buildingPDB-ID: 6DNH

6dnh

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00413768
ELECTRON MICROSCOPYf_angle_d0.59818689
ELECTRON MICROSCOPYf_dihedral_angle_d6.2621878
ELECTRON MICROSCOPYf_chiral_restr0.0452078
ELECTRON MICROSCOPYf_plane_restr0.0052362

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