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- PDB-8r8r: Cryo-EM structure of the human mPSF with PAPOA C-terminus peptide... -

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Basic information

Entry
Database: PDB / ID: 8r8r
TitleCryo-EM structure of the human mPSF with PAPOA C-terminus peptide (PAPOAc)
Components
  • (Cleavage and polyadenylation specificity factor subunit ...) x 2
  • RNA (5'-R(P*AP*AP*UP*AP*AP*A)-3')
  • cDNA FLJ50397, highly similar to Poly(A) polymerase alpha
  • pre-mRNA 3' end processing protein WDR33
KeywordsRNA BINDING PROTEIN / 3-end processing / polyadenylation / PAPOA / pre-mRNA / cleavage and polyadenylation
Function / homology
Function and homology information


co-transcriptional RNA 3'-end processing, cleavage and polyadenylation pathway / Inhibition of Host mRNA Processing and RNA Silencing / RNA 3'-end processing / 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 ...co-transcriptional RNA 3'-end processing, cleavage and polyadenylation pathway / Inhibition of Host mRNA Processing and RNA Silencing / RNA 3'-end processing / 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 / nucleotidyltransferase activity / fibrillar center / mRNA processing / sequence-specific double-stranded DNA binding / spermatogenesis / intracellular membrane-bounded organelle / enzyme binding / RNA binding / zinc ion binding / nucleoplasm / ATP binding / 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 / Nucleotidyltransferase, class I-like, C-terminal / 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. ...Zinc-finger CCCH domain / Zinc-finger containing family / CPSF complex subunit CPSF4-like / Pre-mRNA 3' end processing protein Pfs2-like / Nucleotidyltransferase, class I-like, C-terminal / 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 / cDNA FLJ50397, highly similar to Poly(A) polymerase alpha / 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: 2.79 Å
AuthorsTodesca, S. / Sandmeir, F. / Keidel, A. / Conti, E.
Funding support Germany, European Union, Denmark, 4items
OrganizationGrant numberCountry
Max Planck Society Germany
European Research Council (ERC)101054447European Union
German Research Foundation (DFG)SFB1035 Germany
Novo Nordisk Foundation31199 Denmark
CitationJournal: RNA / Year: 2024
Title: Molecular basis of human polyA polymerase recruitment by mPSF.
Authors: Sofia Todesca / Felix Sandmeir / Achim Keidel / Elena Conti
Abstract: 3' end processing of most eukaryotic pre-mRNAs is a crucial co-transcriptional process that generally involves the cleavage and polyadenylation of the precursor transcripts. Within the human 3' end ...3' end processing of most eukaryotic pre-mRNAs is a crucial co-transcriptional process that generally involves the cleavage and polyadenylation of the precursor transcripts. Within the human 3' end processing machinery, the 4-subunit mammalian polyadenylation specificity factor (mPSF) recognizes the polyadenylation signal (PAS) in the pre-mRNA and recruits the polyA polymerase α (PAPOA) to it. To shed light on the molecular mechanisms of PAPOA recruitment to mPSF, we used a combination of cryogenic-electron microscopy (cryo-EM) single-particle analysis, computational structure prediction and biochemistry to reveal an intricate interaction network. A short linear motif in the mPSF subunit FIP1 interacts with the structured core of human PAPOA, with a binding mode that is evolutionary conserved from yeast to human. In higher eukaryotes, however, PAPOA contains a conserved C-terminal motif that can interact intramolecularly with the same residues of the PAPOA structured core used to bind FIP1. Interestingly, using biochemical assay and cryo-EM structural analysis, we found that the PAPOA C-terminal motif can also directly interact with mPSF at the subunit CPSF160. These results show that PAPOA recruitment to mPSF is mediated by two distinct intermolecular connections and further suggest the presence of mutually exclusive interactions in the regulation of 3' end processing.
History
DepositionNov 29, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 10, 2024Provider: 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: Cleavage and polyadenylation specificity factor subunit 4
D: cDNA FLJ50397, highly similar to Poly(A) polymerase alpha
E: RNA (5'-R(P*AP*AP*UP*AP*AP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)245,3648
Polymers245,1675
Non-polymers1963
Water0
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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Cleavage and polyadenylation specificity factor subunit ... , 2 types, 2 molecules AC

#1: Protein Cleavage and polyadenylation specificity factor subunit 1 /


Mass: 161074.234 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CPSF1 / Production host: Homo sapiens (human) / References: UniProt: Q10570
#3: Protein Cleavage and polyadenylation specificity factor subunit 4 /


Mass: 31733.426 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CPSF4 / Production host: Homo sapiens (human) / References: UniProt: O95639

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Protein / Protein/peptide / RNA chain / Non-polymers , 4 types, 6 molecules BDE

#2: Protein pre-mRNA 3' end processing protein WDR33


Mass: 47580.129 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: WDR33 / Production host: Homo sapiens (human) / References: UniProt: Q9C0J8
#4: Protein/peptide cDNA FLJ50397, highly similar to Poly(A) polymerase alpha


Mass: 2872.410 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: B4DZL2
#5: RNA chain RNA (5'-R(P*AP*AP*UP*AP*AP*A)-3')


Mass: 1907.237 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Homo sapiens (human)
#6: 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

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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: mPSF with PAPOA C-terminus peptide (PAPOAc) / Type: COMPLEX / Entity ID: #1-#5 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.9
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 60.6 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.79 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 186241 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00213433
ELECTRON MICROSCOPYf_angle_d0.50918303
ELECTRON MICROSCOPYf_dihedral_angle_d5.4281877
ELECTRON MICROSCOPYf_chiral_restr0.0442085
ELECTRON MICROSCOPYf_plane_restr0.0042307

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