National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R00GM132544
United States
Citation
Journal: bioRxiv / Year: 2025 Title: RNA-induced PRC2 inhibition depends on the sequence of bound RNA. Authors: Jiarui Song / Liqi Yao / Anne R Gooding / Valentin Thron / Wayne O Hemphill / Karen Goodrich / Vignesh Kasinath / Thomas R Cech Abstract: Methyltransferase PRC2 (Polycomb Repressive Complex 2) deposits histone H3K27 trimethylation to establish and maintain epigenetic gene silencing. PRC2 is precisely regulated by accessory proteins, ...Methyltransferase PRC2 (Polycomb Repressive Complex 2) deposits histone H3K27 trimethylation to establish and maintain epigenetic gene silencing. PRC2 is precisely regulated by accessory proteins, histone post-translational modifications and, particularly, 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, a mechanism explained by our recent cryo-EM structure showing G4 RNA-mediated PRC2 dimerization. However, PRC2 binds a wide variety of RNA sequences, and it remains unclear how diverse RNAs beyond G4 associate with and regulate PRC2. Here, we show that variations in RNA sequence elicit distinct effects on PRC2 function. A single-stranded G-rich RNA and an atypical G4 structure called a pUG-fold mediate PRC2 dimerization nearly identical to that induced by G4 RNA. In contrast, pyrimidine-rich RNAs, including a motif identified by CLIPseq in cells, do not induce PRC2 dimerization and instead bind PRC2 monomers with retention of methyltransferase activity. Only RNAs that dimerize PRC2 compete with nucleosome binding and inhibit PRC2 methyltransferase activity. CRISPR-dCas9 was adapted to localize different RNA elements onto a PRC2-targeted gene, revealing RNA sequence specificity for PRC2 regulation in cells. Thus, PRC2 binds many different RNAs with similar affinity, however, the functional effect on enzymatic activity depends entirely on the sequence of the bound RNA, a conclusion potentially applicable to any RNA- binding protein with a large transcriptome.
Name: ZINC ION / type: ligand / ID: 8 / Number of copies: 14 / Formula: ZN
Molecular weight
Theoretical: 65.409 Da
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Buffer
pH: 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).
Vitrification
Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 281 K / Instrument: LEICA EM GP / Details: 2-3s of single side blotting.
Details
We used streptavidin-affinity grid preparation method with biotin-labeled RNA at 100 nM concentration. PRC2 was applied in excess at 600 nM.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recording
Film or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 2 / Number real images: 14230 / Average electron dose: 50.0 e/Å2 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.
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Chain - Source name: PDB / Chain - Initial model type: experimental model
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.
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Movie
Controller
Open data
Basic information
Map data
Sample
Keywords
Function and homology information
Homo sapiens (human)
Authors
United States, 2 items 
Citation
Structure visualization
Downloads & links
emd_46751.png
http://ftp.pdbj.org/pub/emdb/structures/EMD-46751
Z (Sec.)
Y (Row.)
X (Col.)
Sample components
Trichoplusia ni (cabbage looper)
Processing
Electron microscopy
FIELD EMISSION GUN
