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- EMDB-19008: Cryo-EM structure of the human mPSF with PAPOA C-terminus peptide... -

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Entry
Database: EMDB / ID: EMD-19008
TitleCryo-EM structure of the human mPSF with PAPOA C-terminus peptide (PAPOAc)
Map data
Sample
  • Complex: mPSF with PAPOA C-terminus peptide (PAPOAc)
    • Protein or peptide: Cleavage and polyadenylation specificity factor subunit 1
    • Protein or peptide: pre-mRNA 3' end processing protein WDR33
    • Protein or peptide: Cleavage and polyadenylation specificity factor subunit 4
    • Protein or peptide: cDNA FLJ50397, highly similar to Poly(A) polymerase alpha
    • RNA: RNA (5'-R(P*AP*AP*UP*AP*AP*A)-3')
  • Ligand: ZINC ION
Keywords3-end processing / polyadenylation / PAPOA / pre-mRNA / cleavage and polyadenylation / RNA BINDING PROTEIN
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
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.79 Å
AuthorsTodesca S / Sandmeir F / Keidel A / Conti E
Funding support Germany, European Union, Denmark, 4 items
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, 2023-
Header (metadata) releaseApr 10, 2024-
Map releaseApr 10, 2024-
UpdateApr 10, 2024-
Current statusApr 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19008.png

Downloads & links

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Map

FileDownload / File: emd_19008.map.gz / Format: CCP4 / Size: 85.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.8512 Å
Density
Contour LevelBy AUTHOR: 0.337
Minimum - Maximum-0.42857897 - 1.4527484
Average (Standard dev.)0.007873362 (±0.06757466)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions282282282
Spacing282282282
CellA=B=C: 240.03839 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: sharpened map - B factor 116.4

Fileemd_19008_additional_1.map
Annotationsharpened map - B factor 116.4
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: deepEMhancer map

Fileemd_19008_additional_2.map
AnnotationdeepEMhancer map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_19008_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_19008_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : mPSF with PAPOA C-terminus peptide (PAPOAc)

EntireName: mPSF with PAPOA C-terminus peptide (PAPOAc)
Components
  • Complex: mPSF with PAPOA C-terminus peptide (PAPOAc)
    • Protein or peptide: Cleavage and polyadenylation specificity factor subunit 1
    • Protein or peptide: pre-mRNA 3' end processing protein WDR33
    • Protein or peptide: Cleavage and polyadenylation specificity factor subunit 4
    • Protein or peptide: cDNA FLJ50397, highly similar to Poly(A) polymerase alpha
    • RNA: RNA (5'-R(P*AP*AP*UP*AP*AP*A)-3')
  • Ligand: ZINC ION

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Supramolecule #1: mPSF with PAPOA C-terminus peptide (PAPOAc)

SupramoleculeName: mPSF with PAPOA C-terminus peptide (PAPOAc) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Cleavage and polyadenylation specificity factor subunit 1

MacromoleculeName: Cleavage and polyadenylation specificity factor subunit 1
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 161.074234 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MYAVYKQAHP PTGLEFSMYC NFFNNSERNL VVAGTSQLYV YRLNRDAEAL TKNDRSTEGK AHREKLELAA SFSFFGNVMS MASVQLAGA KRDALLLSFK DAKLSVVEYD PGTHDLKTLS LHYFEEPELR DGFVQNVHTP RVRVDPDGRC AAMLVYGTRL V VLPFRRES ...String:
MYAVYKQAHP PTGLEFSMYC NFFNNSERNL VVAGTSQLYV YRLNRDAEAL TKNDRSTEGK AHREKLELAA SFSFFGNVMS MASVQLAGA KRDALLLSFK DAKLSVVEYD PGTHDLKTLS LHYFEEPELR DGFVQNVHTP RVRVDPDGRC AAMLVYGTRL V VLPFRRES LAEEHEGLVG EGQRSSFLPS YIIDVRALDE KLLNIIDLQF LHGYYEPTLL ILFEPNQTWP GRVAVRQDTC SI VAISLNI TQKVHPVIWS LTSLPFDCTQ ALAVPKPIGG VVVFAVNSLL YLNQSVPPYG VALNSLTTGT TAFPLRTQEG VRI TLDCAQ ATFISYDKMV ISLKGGEIYV LTLITDGMRS VRAFHFDKAA ASVLTTSMVT MEPGYLFLGS RLGNSLLLKY TEKL QEPPA SAVREAADKE EPPSKKKRVD ATAGWSAAGK SVPQDEVDEI EVYGSEAQSG TQLATYSFEV CDSILNIGPC ANAAV GEPA FLSEEFQNSP EPDLEIVVCS GHGKNGALSV LQKSIRPQVV TTFELPGCYD MWTVIAPVRK EEEDNPKGEG TEQEPS TTP EADDDGRRHG FLILSREDST MILQTGQEIM ELDTSGFATQ GPTVFAGNIG DNRYIVQVSP LGIRLLEGVN QLHFIPV DL GAPIVQCAVA DPYVVIMSAE GHVTMFLLKS DSYGGRHHRL ALHKPPLHHQ SKVITLCLYR DLSGMFTTES RLGGARDE L GGRSGPEAEG LGSETSPTVD DEEEMLYGDS GSLFSPSKEE ARRSSQPPAD RDPAPFRAEP THWCLLVREN GTMEIYQLP DWRLVFLVKN FPVGQRVLVD SSFGQPTTQG EARREEATRQ GELPLVKEVL LVALGSRQSR PYLLVHVDQE LLIYEAFPHD SQLGQGNLK VRFKKVPHNI NFREKKPKPS KKKAEGGGAE EGAGARGRVA RFRYFEDIYG YSGVFICGPS PHWLLVTGRG A LRLHPMAI DGPVDSFAPF HNVNCPRGFL YFNRQGELRI SVLPAYLSYD APWPVRKIPL RCTAHYVAYH VESKVYAVAT ST NTPCARI PRMTGEEKEF ETIERDERYI HPQQEAFSIQ LISPVSWEAI PNARIELQEW EHVTCMKTVS LRSEETVSGL KGY VAAGTC LMQGEEVTCR GRILIMDVIE VVPEPGQPLT KNKFKVLYEK EQKGPVTALC HCNGHLVSAI GQKIFLWSLR ASEL TGMAF IDTQLYIHQM ISVKNFILAA DVMKSISLLR YQEESKTLSL VSRDAKPLEV YSVDFMVDNA QLGFLVSDRD RNLMV YMYL PEAKESFGGM RLLRRADFHV GAHVNTFWRT PCRGATEGLS KKSVVWENKH ITWFATLDGG IGLLLPMQEK TYRRLL MLQ NALTTMLPHH AGLNPRAFRM LHVDRRTLQN AVRNVLDGEL LNRYLYLSTM ERSELAKKIG TTPDIILDDL LETDRVT AH F

UniProtKB: Cleavage and polyadenylation specificity factor subunit 1

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

MacromoleculeName: pre-mRNA 3' end processing protein WDR33 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 47.580129 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MATEIGSPPR FFHMPRFQHQ APRQLFYKRP DFAQQQAMQQ LTFDGKRMRK AVNRKTIDYN PSVIKYLENR IWQRDQRDMR AIQPDAGYY NDLVPPIGML NNPMNAVTTK FVRTSTNKVK CPVFVVRWTP EGRRLVTGAS SGEFTLWNGL TFNFETILQA H DSPVRAMT ...String:
MATEIGSPPR FFHMPRFQHQ APRQLFYKRP DFAQQQAMQQ LTFDGKRMRK AVNRKTIDYN PSVIKYLENR IWQRDQRDMR AIQPDAGYY NDLVPPIGML NNPMNAVTTK FVRTSTNKVK CPVFVVRWTP EGRRLVTGAS SGEFTLWNGL TFNFETILQA H DSPVRAMT WSHNDMWMLT ADHGGYVKYW QSNMNNVKMF QAHKEAIREA SFSPTDNKFA TCSDDGTVRI WDFLRCHEER IL RGHGADV KCVDWHPTKG LVVSGSKDSQ QPIKFWDPKT GQSLATLHAH KNTVMEVKLN LNGNWLLTAS RDHLCKLFDI RNL KEELQV FRGHKKEATA VAWHPVHEGL FASGGSDGSL LFWHVGVEKE VGGMEMAHEG MIWSLAWHPL GHILCSGSND HTSK FWTRN RPGDKMRD

UniProtKB: pre-mRNA 3' end processing protein WDR33

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Macromolecule #3: Cleavage and polyadenylation specificity factor subunit 4

MacromoleculeName: Cleavage and polyadenylation specificity factor subunit 4
type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 31.733426 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSAWSHPQFE KGGGSGGGSG GSAWSHPQFE KTAGLEVLFQ GPQEIIASVD HIKFDLEIAV EQQLGAQPLP FPGMDKSGAA VCEFFLKAA CGKGGMCPFR HISGEKTVVC KHWLRGLCKK GDQCEFLHEY DMTKMPECYF YSKFGECSNK ECPFLHIDPE S KIKDCPWY ...String:
MSAWSHPQFE KGGGSGGGSG GSAWSHPQFE KTAGLEVLFQ GPQEIIASVD HIKFDLEIAV EQQLGAQPLP FPGMDKSGAA VCEFFLKAA CGKGGMCPFR HISGEKTVVC KHWLRGLCKK GDQCEFLHEY DMTKMPECYF YSKFGECSNK ECPFLHIDPE S KIKDCPWY DRGFCKHGPL CRHRHTRRVI CVNYLVGFCP EGPSCKFMHP RFELPMGTTE QPPLPQQTQP PAKQRTPQVI GV MQSQNSS AGNRGPRPLE QVTCYKCGEK GHYANRCTKG HLAFLSGQ

UniProtKB: Cleavage and polyadenylation specificity factor subunit 4

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Macromolecule #4: cDNA FLJ50397, highly similar to Poly(A) polymerase alpha

MacromoleculeName: cDNA FLJ50397, highly similar to Poly(A) polymerase alpha
type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 2.87241 KDa
SequenceString:
DLSDIPALPA NPIPVIKNSI KLRLNR

UniProtKB: cDNA FLJ50397, highly similar to Poly(A) polymerase alpha

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Macromolecule #5: RNA (5'-R(P*AP*AP*UP*AP*AP*A)-3')

MacromoleculeName: RNA (5'-R(P*AP*AP*UP*AP*AP*A)-3') / type: rna / ID: 5 / Number of copies: 1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 1.907237 KDa
SequenceString:
AAUAAA

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 3 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.9
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 105000
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.79 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 186241

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