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- EMDB-14710: Polymerase module of CPF in complex with Mpe1 and a pre-cleaved C... -

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Basic information

Entry
Database: EMDB / ID: EMD-14710
TitlePolymerase module of CPF in complex with Mpe1 and a pre-cleaved CYC1 RNA
Map data
Sample
  • Complex: polymerase module-Mpe1-RNA
    • Protein or peptide: Protein CFT1
    • Protein or peptide: mRNA 3'-end-processing protein YTH1
    • Protein or peptide: Polyadenylation factor subunit 2
    • RNA: pre-cleaved CYC1
    • Protein or peptide: MPE1 isoform 1
  • Ligand: ZINC ION
KeywordsCPF / 3'-end processing / polyA / RNA BINDING PROTEIN
Function / homology
Function and homology information


: / Processing of Intronless Pre-mRNAs / termination of RNA polymerase II transcription, poly(A)-coupled / mRNA cleavage and polyadenylation specificity factor complex / mRNA 3'-end processing / termination of RNA polymerase II transcription / mRNA processing / ubiquitin protein ligase activity / ubiquitin-dependent protein catabolic process / nucleic acid binding ...: / Processing of Intronless Pre-mRNAs / termination of RNA polymerase II transcription, poly(A)-coupled / mRNA cleavage and polyadenylation specificity factor complex / mRNA 3'-end processing / termination of RNA polymerase II transcription / mRNA processing / ubiquitin protein ligase activity / ubiquitin-dependent protein catabolic process / nucleic acid binding / protein ubiquitination / mitochondrion / RNA binding / zinc ion binding / nucleus / metal ion binding
Similarity search - Function
DWNN domain / DWNN domain / DWNN domain profile. / DWNN / CPSF complex subunit CPSF4-like / RNA-binding, Nab2-type zinc finger / 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 ...DWNN domain / DWNN domain / DWNN domain profile. / DWNN / CPSF complex subunit CPSF4-like / RNA-binding, Nab2-type zinc finger / 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. / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / : / CPSF A subunit region / zinc finger / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / Zinc finger, RING/FYVE/PHD-type / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily
Similarity search - Domain/homology
MPE1 isoform 1 / mRNA 3'-end-processing protein / Polyadenylation factor subunit 2 / Protein CFT1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsRodriguez-Molina JB / Passmore LA
Funding supportEuropean Union, United Kingdom, 2 items
OrganizationGrant numberCountry
European Research Council (ERC)725685European Union
Medical Research Council (MRC, United Kingdom)MC_U105192715 United Kingdom
CitationJournal: Mol Cell / Year: 2022
Title: Mpe1 senses the binding of pre-mRNA and controls 3' end processing by CPF.
Authors: Juan B Rodríguez-Molina / Francis J O'Reilly / Holly Fagarasan / Eleanor Sheekey / Sarah Maslen / J Mark Skehel / Juri Rappsilber / Lori A Passmore /
Abstract: Most eukaryotic messenger RNAs (mRNAs) are processed at their 3' end by the cleavage and polyadenylation specificity factor (CPF/CPSF). CPF mediates the endonucleolytic cleavage of the pre-mRNA and ...Most eukaryotic messenger RNAs (mRNAs) are processed at their 3' end by the cleavage and polyadenylation specificity factor (CPF/CPSF). CPF mediates the endonucleolytic cleavage of the pre-mRNA and addition of a polyadenosine (poly(A)) tail, which together define the 3' end of the mature transcript. The activation of CPF is highly regulated to maintain the fidelity of RNA processing. Here, using cryo-EM of yeast CPF, we show that the Mpe1 subunit directly contacts the polyadenylation signal sequence in nascent pre-mRNA. The region of Mpe1 that contacts RNA also promotes the activation of CPF endonuclease activity and controls polyadenylation. The Cft2 subunit of CPF antagonizes the RNA-stabilized configuration of Mpe1. In vivo, the depletion or mutation of Mpe1 leads to widespread defects in transcription termination by RNA polymerase II, resulting in transcription interference on neighboring genes. Together, our data suggest that Mpe1 plays a major role in accurate 3' end processing, activating CPF, and ensuring timely transcription termination.
History
DepositionApr 4, 2022-
Header (metadata) releaseMay 25, 2022-
Map releaseMay 25, 2022-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14710.png

Downloads & links

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Map

FileDownload / File: emd_14710.map.gz / Format: CCP4 / Size: 160.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 348 pix.
= 287.1 Å		0.83 Å/pix.
x 348 pix.
= 287.1 Å		0.83 Å/pix.
x 348 pix.
= 287.1 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.016
Minimum - Maximum-0.03533315 - 0.078095436
Average (Standard dev.)0.000031280204 (±0.002323335)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions348348348
Spacing348348348
CellA=B=C: 287.1 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_14710_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_14710_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 : polymerase module-Mpe1-RNA

EntireName: polymerase module-Mpe1-RNA
Components
  • Complex: polymerase module-Mpe1-RNA
    • Protein or peptide: Protein CFT1
    • Protein or peptide: mRNA 3'-end-processing protein YTH1
    • Protein or peptide: Polyadenylation factor subunit 2
    • RNA: pre-cleaved CYC1
    • Protein or peptide: MPE1 isoform 1
  • Ligand: ZINC ION

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Supramolecule #1: polymerase module-Mpe1-RNA

SupramoleculeName: polymerase module-Mpe1-RNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: Protein CFT1

MacromoleculeName: Protein CFT1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 153.577156 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MNVYDDVLDA TVVSHSLATH FTTSDYEELL VVRTNILSVY RPTRDGKLYL TDEFKFHGLI TDIGLIPQKD SPLSCLLLCT GVAKISILK FNTLTNSIDT LSLHYYEGKF KGKSLVELAK ISTLRMDPGS SCALLFNNDI IAFLPFHVNK NDDDEEEEDE D ENIDDSEL ...String:
MNVYDDVLDA TVVSHSLATH FTTSDYEELL VVRTNILSVY RPTRDGKLYL TDEFKFHGLI TDIGLIPQKD SPLSCLLLCT GVAKISILK FNTLTNSIDT LSLHYYEGKF KGKSLVELAK ISTLRMDPGS SCALLFNNDI IAFLPFHVNK NDDDEEEEDE D ENIDDSEL IHSMNQKSQG TNTFNKRKRT KLGDKFTAPS VVLVASELYE GAKNIIDIQF LKNFTKPTIA LLYQPKLVWA GN TTISKLP TQYVILTLNI QPAESATKIE STTIAFVKEL PWDLHTIVPV SNGAIIVGTN ELAFLDNTGV LQSTVLLNSF ADK ELQKTK IINNSSLEIM FREKNTTSIW IPSSKSKNGG SNNDETLLLM DLKSNIYYIQ MEAEGRLLIK FDIFKLPIVN DLLK ENSNP KCITRLNATN SNKNMDLFIG FGSGNALVLR LNNLKSTIET REAHNPSSGT NSLMDINDDD DEEMDDLYAD EAPEN GLTT NDSKGTVETV QPFDIELLSS LRNVGPITSL TVGKVSSIDD VVKGLPNPNK NEYSLVATSG NGSGSHLTVI QTSVQP EIE LALKFISITQ IWNLKIKGRD RYLITTDSTK SRSDIYESDN NFKLHKGGRL RRDATTVYIS MFGEEKRIIQ VTTNHLY LY DTHFRRLTTI KFDYEVIHVS VMDPYILVTV SRGDIKIFEL EEKNKRKLLK VDLPEILNEM VITSGLILKS NMCNEFLI G LSKSQEEQLL FTFVTADNQI IFFTKDHNDR IFQLNGVDQL NESLYISTYQ LGDEIVPDPS IKQVMINKLG HDNKEEYLT ILTFGGEIYQ YRKLPQRRSR FYRNVTRNDL AITGAPDNAY AKGVSSIERI MHYFPDYNGY SVIFVTGSVP YILIKEDDST PKIFKFGNI PLVSVTPWSE RSVMCVDDIK NARVYTLTTD NMYYGNKLPL KQIKISNVLD DYKTLQKLVY HERAQLFLVS Y CKRVPYEA LGEDGEKVIG YDENVPHAEG FQSGILLINP KSWKVIDKID FPKNSVVNEM RSSMIQINSK TKRKREYIIA GV ANATTED TPPTGAFHIY DVIEVVPEPG KPDTNYKLKE IFQEEVSGTV STVCEVSGRF MISQSQKVLV RDIQEDNSVI PVA FLDIPV FVTDSKSFGN LLIIGDAMQG FQFIGFDAEP YRMISLGRSM SKFQTMSLEF LVNGGDMYFA ATDADRNVHV LKYA PDEPN SLSGQRLVHC SSFTLHSTNS CMMLLPRNEE FGSPQVPSFQ NVGGQVDGSV FKIVPLSEEK YRRLYVIQQQ IIDRE LQLG GLNPRMERLA NDFYQMGHSM RPMLDFNVIR RFCGLAIDRR KSIAQKAGRH AHFEAWRDII NIEFSMRSLC QGK

UniProtKB: Protein CFT1

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Macromolecule #2: mRNA 3'-end-processing protein YTH1

MacromoleculeName: mRNA 3'-end-processing protein YTH1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 24.594498 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSLIHPDTAK YPFKFEPFLR QEYSFSLDPD RPICEFYNSR EGPKSCPRGP LCPKKHVLPI FQNKIVCRHW LRGLCKKNDQ CEYLHEYNL RKMPECVFFS KNGYCTQSPD CQYLHIDPAS KIPKCENYEM GFCPLGSSCP RRHIKKVFCQ RYMTGFCPLG K DECDMEHP ...String:
MSLIHPDTAK YPFKFEPFLR QEYSFSLDPD RPICEFYNSR EGPKSCPRGP LCPKKHVLPI FQNKIVCRHW LRGLCKKNDQ CEYLHEYNL RKMPECVFFS KNGYCTQSPD CQYLHIDPAS KIPKCENYEM GFCPLGSSCP RRHIKKVFCQ RYMTGFCPLG K DECDMEHP QFIIPDEGSK LRIKRDDEIN TRKMDEEKER RLNAIINGEV

UniProtKB: mRNA 3'-end-processing protein

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Macromolecule #3: Polyadenylation factor subunit 2

MacromoleculeName: Polyadenylation factor subunit 2 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 53.211117 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDGHNQNQYQ NQNQIQQSQQ PPLKKYVTQR RSVDVSSPYI NLYYNRRHGL PNLVVEPETS YTIDIMPPNA YRGRDRVINL PSKFTHLSS NKVKHVIPAI QWTPEGRRLV VATYSGEFSL WNASSFTFET LMQAHDSAVT TMKYSHDSDW MISGDADGMI K IWQPNFSM ...String:
MDGHNQNQYQ NQNQIQQSQQ PPLKKYVTQR RSVDVSSPYI NLYYNRRHGL PNLVVEPETS YTIDIMPPNA YRGRDRVINL PSKFTHLSS NKVKHVIPAI QWTPEGRRLV VATYSGEFSL WNASSFTFET LMQAHDSAVT TMKYSHDSDW MISGDADGMI K IWQPNFSM VKEIDAAHTE SIRDMAFSSN DSKFVTCSDD NILKIWNFSN GKQERVLSGH HWDVKSCDWH PEMGLIASAS KD NLVKLWD PRSGNCISSI LKFKHTVLKT RFQPTKGNLL MAISKDKSCR VFDIRYSMKE LMCVRDETDY MTLEWHPINE SMF TLACYD GSLKHFDLLQ NLNEPILTIP YAHDKCITSL SYNPVGHIFA TAAKDRTIRF WTRARPIDPN AYDDPTYNNK KING WFFGI NNDINAVREK SEFGAAPPPP ATLEPHALPN MNGFINKKPR QEIPGIDSNI KSSTLPGLSI

UniProtKB: Polyadenylation factor subunit 2

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Macromolecule #5: MPE1 isoform 1

MacromoleculeName: MPE1 isoform 1 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 49.711848 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSSTIFYRFK SQRNTSRILF DGTGLTVFDL KREIIQENKL GDGTDFQLKI YNPDTEEEYD DDAFVIPRST SVIVKRSPAI KSFSVHSRL KGNVGAAALG NATRYVTGRP RVLQKRQHTA TTTANVSGTT EEERIASMFA TQENQWEQTQ EEMSAATPVF F KSQTNKNS ...String:
MSSTIFYRFK SQRNTSRILF DGTGLTVFDL KREIIQENKL GDGTDFQLKI YNPDTEEEYD DDAFVIPRST SVIVKRSPAI KSFSVHSRL KGNVGAAALG NATRYVTGRP RVLQKRQHTA TTTANVSGTT EEERIASMFA TQENQWEQTQ EEMSAATPVF F KSQTNKNS AQENEGPPPP GYMCYRCGGR DHWIKNCPTN SDPNFEGKRI RRTTGIPKKF LKSIEIDPET MTPEEMAQRK IM ITDEGKF VVQVEDKQSW EDYQRKRENR QIDGDETIWR KGHFKDLPDD LKCPLTGGLL RQPVKTSKCC NIDFSKEALE NAL VESDFV CPNCETRDIL LDSLVPDQDK EKEVETFLKK QEELHGSSKD GNQPETKKMK LMDPTGTAGL NNNTSLPTSV NNGG TPVPP VPLPFGIPPF PMFPMPFMPP TATITNPHQA DASPKK

UniProtKB: MPE1 isoform 1

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Macromolecule #4: pre-cleaved CYC1

MacromoleculeName: pre-cleaved CYC1 / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 13.329796 KDa
SequenceString:
UUUAUAGUUA UGUUAGUAUU AAGAACGUUA UUUAUAUUUC AA

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 2 / 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: 8 / Details: 20 mM HEPES pH 8, 50 mM NaCl, 0.5 mM TCEP
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 11856 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.1 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 6460073
Startup modelType of model: EMDB MAP
EMDB ID:

3908

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 131152
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)

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Atomic model buiding 1

Initial modelPDB ID:

6eoj


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-7zgp:
Polymerase module of CPF in complex with Mpe1 and a pre-cleaved CYC1 RNA

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