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- PDB-9dch: Single-stranded RNA-mediated PRC2 dimer -

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Basic information

Entry
Database: PDB / ID: 9dch
TitleSingle-stranded RNA-mediated PRC2 dimer
Components
  • (Polycomb protein ...) x 2
  • Isoform 2 of Histone-lysine N-methyltransferase EZH2
  • Protein Jumonji
  • RBAP48
  • TERRAmut RNA
  • Zinc finger protein AEBP2
KeywordsGENE REGULATION / PRC2 / RNA / RNP complex / chromatin modifier
Function / homology
Function and homology information


protein localization to pericentric heterochromatin / hepatocyte homeostasis / cellular response to trichostatin A / regulation of gliogenesis / negative regulation of striated muscle cell differentiation / regulation of kidney development / [histone H3]-lysine27 N-trimethyltransferase / sex chromatin / CAF-1 complex / negative regulation of keratinocyte differentiation ...protein localization to pericentric heterochromatin / hepatocyte homeostasis / cellular response to trichostatin A / regulation of gliogenesis / negative regulation of striated muscle cell differentiation / regulation of kidney development / [histone H3]-lysine27 N-trimethyltransferase / sex chromatin / CAF-1 complex / negative regulation of keratinocyte differentiation / histone H3K27 trimethyltransferase activity / negative regulation of retinoic acid receptor signaling pathway / response to tetrachloromethane / cerebellar cortex development / primary miRNA binding / random inactivation of X chromosome / regulatory ncRNA-mediated heterochromatin formation / skeletal muscle satellite cell maintenance involved in skeletal muscle regeneration / histone H3K27 methyltransferase activity / negative regulation of cardiac muscle cell proliferation / ubiquitin-modified histone reader activity / facultative heterochromatin formation / positive regulation of cell cycle G1/S phase transition / negative regulation of cardiac muscle hypertrophy / NURF complex / NuRD complex / regulation of cell fate specification / negative regulation of stem cell population maintenance / DNA replication-dependent chromatin assembly / ESC/E(Z) complex / Transcription of E2F targets under negative control by p107 (RBL1) and p130 (RBL2) in complex with HDAC1 / chromatin silencing complex / RSC-type complex / negative regulation of stem cell differentiation / protein-lysine N-methyltransferase activity / regulation of stem cell differentiation / pronucleus / Polo-like kinase mediated events / Transcription of E2F targets under negative control by DREAM complex / cardiac muscle hypertrophy in response to stress / synaptic transmission, GABAergic / G1 to G0 transition / positive regulation of dendrite development / cardiac muscle cell proliferation / histone H3 methyltransferase activity / lncRNA binding / histone methyltransferase complex / DNA methylation-dependent constitutive heterochromatin formation / histone methyltransferase activity / negative regulation of G1/S transition of mitotic cell cycle / spinal cord development / negative regulation of gene expression, epigenetic / ATPase complex / positive regulation of stem cell population maintenance / G1/S-Specific Transcription / Sin3-type complex / Transcriptional Regulation by E2F6 / oligodendrocyte differentiation / negative regulation of transcription elongation by RNA polymerase II / RNA Polymerase I Transcription Initiation / histone deacetylase complex / G0 and Early G1 / negative regulation of cell differentiation / negative regulation of cytokine production involved in inflammatory response / subtelomeric heterochromatin formation / pericentric heterochromatin / RNA polymerase II core promoter sequence-specific DNA binding / ribonucleoprotein complex binding / Transcriptional regulation of brown and beige adipocyte differentiation by EBF2 / positive regulation of epithelial to mesenchymal transition / Cyclin E associated events during G1/S transition / nucleosome binding / Cyclin A:Cdk2-associated events at S phase entry / keratinocyte differentiation / spleen development / Regulation of TP53 Activity through Acetylation / protein localization to chromatin / Deposition of new CENPA-containing nucleosomes at the centromere / methylated histone binding / B cell differentiation / enzyme activator activity / positive regulation of GTPase activity / cellular response to leukemia inhibitory factor / positive regulation of MAP kinase activity / SUMOylation of chromatin organization proteins / liver development / liver regeneration / negative regulation of cell migration / thymus development / hippocampus development / central nervous system development / ubiquitin binding / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / PRC2 methylates histones and DNA / Regulation of PTEN gene transcription / transcription corepressor binding / Regulation of endogenous retroelements by KRAB-ZFP proteins / Defective pyroptosis / positive regulation of protein serine/threonine kinase activity / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs)
Similarity search - Function
: / EZH2, SET domain / Polycomb protein, VEFS-Box / VEFS-Box of polycomb protein / Histone-lysine N-methyltransferase EZH1/EZH2 / Polycomb repressive complex 2 subunit EZH1/EZH2, tri-helical domain / Pre-SET CXC domain / : / WD repeat binding protein EZH2 / Polycomb repressive complex 2 tri-helical domain ...: / EZH2, SET domain / Polycomb protein, VEFS-Box / VEFS-Box of polycomb protein / Histone-lysine N-methyltransferase EZH1/EZH2 / Polycomb repressive complex 2 subunit EZH1/EZH2, tri-helical domain / Pre-SET CXC domain / : / WD repeat binding protein EZH2 / Polycomb repressive complex 2 tri-helical domain / CXC domain / Ezh2, MCSS domain / Tesmin/TSO1-like CXC domain / Tesmin/TSO1-like CXC domain / CXC domain / Histone-lysine N-methyltransferase EZH1/2-like / CXC domain profile. / Histone-binding protein RBBP4, N-terminal / Histone-binding protein RBBP4 or subunit C of CAF1 complex / : / : / Zinc finger, C5HC2-type / C5HC2 zinc finger / ARID DNA-binding domain / ARID DNA-binding domain superfamily / ARID/BRIGHT DNA binding domain / ARID domain profile. / BRIGHT, ARID (A/T-rich interaction domain) domain / ARID/BRIGHT DNA binding domain / JmjN domain / jmjN domain / JmjN domain profile. / Small domain found in the jumonji family of transcription factors / SET (Su(var)3-9, Enhancer-of-zeste, Trithorax) domain / SET domain / SET domain superfamily / SET domain profile. / SET domain / JmjC domain, hydroxylase / SANT SWI3, ADA2, N-CoR and TFIIIB'' DNA-binding domains / A domain family that is part of the cupin metalloenzyme superfamily. / SANT/Myb domain / JmjC domain / JmjC domain profile. / zinc finger / Zinc finger C2H2 type domain profile. / Zinc finger C2H2 superfamily / Zinc finger C2H2 type domain signature. / Zinc finger C2H2-type / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / 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
RNA / Polycomb protein EED / Histone-binding protein RBBP4 / Polycomb protein SUZ12 / Histone-lysine N-methyltransferase EZH2 / Zinc finger protein AEBP2 / Protein Jumonji
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsSong, J.S. / Kasinath, V.K.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00GM132544 United States
CitationJournal: bioRxiv / Year: 2024
Title: Diverse RNA Structures Induce PRC2 Dimerization and Inhibit Histone Methyltransferase Activity.
Authors: Jiarui Song / Liqi Yao / Anne R Gooding / Valentin Thron / Vignesh Kasinath / Thomas R Cech
Abstract: Methyltransferase PRC2 (Polycomb Repressive Complex 2) introduces histone H3K27 trimethylation, a repressive chromatin mark, to tune the differential expression of genes. PRC2 is precisely regulated ...Methyltransferase PRC2 (Polycomb Repressive Complex 2) introduces histone H3K27 trimethylation, a repressive chromatin mark, to tune the differential expression of genes. PRC2 is precisely regulated by accessory proteins, histone post-translational modifications and, notably, RNA. Research on PRC2-associated RNA has mostly focused on the tight-binding G-quadruplex (G4) RNAs, which inhibit PRC2 enzymatic activity in vitro and in cells. Our recent cryo-EM structure provided a molecular mechanism for G4 RNA inactivating PRC2 via dimerization, but it remained unclear how diverse RNAs associate with and regulate PRC2. Here, we show that a single-stranded G-rich RNA and an atypical G4 structure called pUG-fold unexpectedly also mediate near-identical PRC2 dimerization resulting in inhibition of PRC2 methyltransferase activity. The conformational flexibility of arginine-rich loops within subunits EZH2 and AEBP2 of PRC2 can accommodate diverse RNA secondary structures, resulting in protein-RNA and protein-protein interfaces similar to those observed previously with G4 RNA. Furthermore, we address a recent report that failed to detect PRC2-associated RNAs in living cells by demonstrating the insensitivity of PRC2-RNA interaction to photochemical crosslinking. Our results support the significance of RNA-mediated PRC2 regulation by showing that this interaction is not limited to a single RNA secondary structure, consistent with the broad PRC2 transcriptome containing many G-tract RNAs incapable of folding into G4 structures.
History
DepositionAug 26, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 12, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
G: TERRAmut RNA
A: Isoform 2 of Histone-lysine N-methyltransferase EZH2
B: Polycomb protein SUZ12
D: RBAP48
F: Zinc finger protein AEBP2
E: Protein Jumonji
C: Polycomb protein EED
H: Isoform 2 of Histone-lysine N-methyltransferase EZH2
I: Polycomb protein SUZ12
K: RBAP48
M: Zinc finger protein AEBP2
L: Protein Jumonji
J: Polycomb protein EED
hetero molecules


Theoretical massNumber of molelcules
Total (without water)674,67627
Polymers673,76013
Non-polymers91614
Water00
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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Protein , 4 types, 8 molecules AHDKFMEL

#2: Protein Isoform 2 of Histone-lysine N-methyltransferase EZH2 / ENX-1 / Enhancer of zeste homolog 2 / Lysine N-methyltransferase 6


Mass: 86017.859 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EZH2, KMT6 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: Q15910, [histone H3]-lysine27 N-trimethyltransferase
#4: Protein RBAP48 / Chromatin assembly factor 1 subunit C / CAF-1 subunit C / Chromatin assembly factor I p48 subunit / ...Chromatin assembly factor 1 subunit C / CAF-1 subunit C / Chromatin assembly factor I p48 subunit / CAF-I 48 kDa subunit / CAF-I p48 / Nucleosome-remodeling factor subunit RBAP48 / Retinoblastoma-binding protein 4 / RBBP-4 / Retinoblastoma-binding protein p48


Mass: 47709.527 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RBBP4, RBAP48 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q09028
#5: Protein Zinc finger protein AEBP2 / Adipocyte enhancer-binding protein 2 / AE-binding protein 2


Mass: 32881.473 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AEBP2 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q6ZN18
#6: Protein Protein Jumonji / Jumonji/ARID domain-containing protein 2


Mass: 36021.391 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: JARID2, JMJ / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q92833

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Polycomb protein ... , 2 types, 4 molecules BICJ

#3: Protein Polycomb protein SUZ12 / Chromatin precipitated E2F target 9 protein / ChET 9 protein / Joined to JAZF1 protein / Suppressor ...Chromatin precipitated E2F target 9 protein / ChET 9 protein / Joined to JAZF1 protein / Suppressor of zeste 12 protein homolog


Mass: 82353.133 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SUZ12, CHET9, JJAZ1, KIAA0160 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q15022
#7: Protein Polycomb protein EED / hEED / Embryonic ectoderm development protein / WD protein associating with integrin cytoplasmic ...hEED / Embryonic ectoderm development protein / WD protein associating with integrin cytoplasmic tails 1 / WAIT-1


Mass: 50267.691 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EED / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O75530

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RNA chain / Non-polymers , 2 types, 15 molecules G

#1: RNA chain TERRAmut RNA


Mass: 3257.980 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#8: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN
Has protein modificationY

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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

Component
IDNameTypeEntity IDParent-IDSource
1Single-stranded RNA-mediated dimer of Polycomb Repressive Complex 2COMPLEX#1-#70MULTIPLE SOURCES
2dimer of Polycomb Repressive Complex 2COMPLEX#2-#71RECOMBINANT
3TERRAmut RNACOMPLEX#11SYNTHETIC
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)Organism: Trichoplusia ni (cabbage looper) / Strain: BTI-Tn-5B1-4
Buffer solutionpH: 7.9
Details: RNP complex buffer (25 mM HEPES pH 7.9, 50 mM KCl, 2 mM MgCl2, 10% glycerol, and 1 mM TCEP) EM preparation buffer I (25 mM HEPES pH 7.9, 50 mM KCl, 2.5% glycerol, and 1 mM TCEP) EM ...Details: RNP complex buffer (25 mM HEPES pH 7.9, 50 mM KCl, 2 mM MgCl2, 10% glycerol, and 1 mM TCEP) EM preparation buffer I (25 mM HEPES pH 7.9, 50 mM KCl, 2.5% glycerol, and 1 mM TCEP) EM preparation buffer II (25 mM HEPES pH 7.9, 50 mM KCl, 2.5% glycerol, 0.01%NP-40, and 1 mM TCEP).
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: We used streptavidin-affinity grid preparation method with biotin-labeled RNA at 100 nM concentration. PRC2 was applied in excess at 600 nM.
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 281 K / Details: 2-3s of single side blotting

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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 FIELD / Calibrated magnification: 130000 X / Nominal defocus max: 1900 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 14230
Details: Cryo-EM data was collected using a Titan Krios G3i equipped with a Thermo Fisher Falcon 4 direct-electron detector (DED) camera and a Selectris energy filter set with a 10-eV slit width. ...Details: Cryo-EM data was collected using a Titan Krios G3i equipped with a Thermo Fisher Falcon 4 direct-electron detector (DED) camera and a Selectris energy filter set with a 10-eV slit width. Data acquisition was performed using Thermo Fisher EPU at 130,000x magnification (0.97 A/pixel) with a defocus range of minus 1.9 to minus 0.5 micrometer. Movies were collected in EER format with a total dose of 50 electrons per square angstrom and an exposure time of 5.49 s corresponding to 1323 frames.
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1RELION5particle selection
2EPUimage acquisition
4RELION5CTF correction
7Cootmodel fitting
9RELION5initial Euler assignment
10RELION5final Euler assignment
11RELION5classification
12RELION53D reconstruction
13PHENIXmodel refinement
Image processingDetails: Gain correction was applied during motion correction using Relion own implementation of the UCSF motioncor2 program
CTF correctionDetails: CTFind / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3385491 / Details: TOPAZ auto picking
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 120658 / Symmetry type: POINT
Atomic model buildingDetails: Individual PRC2 protomers were built using cryo-EM maps from the multibody refinement. The coordinates of G-quadruplex RNA-bound PRC2 six-subunit complex (PDB: 8FYH) provided a starting ...Details: Individual PRC2 protomers were built using cryo-EM maps from the multibody refinement. The coordinates of G-quadruplex RNA-bound PRC2 six-subunit complex (PDB: 8FYH) provided a starting model from which all the coordinates were adjusted and rebuilt in the new map using COOT. The model of each PRC2 promoter was subjected to global refinement and minimization in real space using PHENIX. These were then subjected to manual inspection and adjustment in COOT, followed by refinement again in PHENIX. TERRAmut RNA model was generated using a 10-nucleotide single-stranded fragment (UGAGUGUGAG) using AlphaFold3 and then docked into our map for the position we designated as the RNA density.
Atomic model buildingPDB-ID: 8FYH

8fyh


Accession code: 8FYH / Source name: PDB / Type: experimental model

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