National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R00GM132544
米国
引用
ジャーナル: bioRxiv / 年: 2024 タイトル: Diverse RNA Structures Induce PRC2 Dimerization and Inhibit Histone Methyltransferase Activity. 著者: Jiarui Song / Liqi Yao / Anne R Gooding / Valentin Thron / Vignesh Kasinath / Thomas R Cech 要旨: 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.
pH: 7.9 詳細: 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 ...詳細: 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).
凍結
凍結剤: ETHANE / チャンバー内湿度: 90 % / チャンバー内温度: 281 K / 装置: LEICA EM GP / 詳細: 2-3s of single side blotting.
詳細
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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電子顕微鏡法
顕微鏡
FEI TITAN KRIOS
特殊光学系
エネルギーフィルター - 名称: TFS Selectris X / エネルギーフィルター - スリット幅: 10 eV
撮影
フィルム・検出器のモデル: FEI FALCON IV (4k x 4k) 撮影したグリッド数: 2 / 実像数: 14230 / 平均電子線量: 50.0 e/Å2 詳細: 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 ...詳細: 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.
Chain - Source name: PDB / Chain - Initial model type: experimental model
詳細
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.
ムービー
コントローラー
データを開く
基本情報
マップデータ
試料
キーワード
機能・相同性情報
Homo sapiens (ヒト)
データ登録者
米国, 2件 
引用
構造の表示
ダウンロードとリンク
emd_46751.png
http://ftp.pdbj.org/pub/emdb/structures/EMD-46751
Z (Sec.)
Y (Row.)
X (Col.)
試料の構成要素
Trichoplusia ni (イラクサキンウワバ)
解析
電子顕微鏡法
FIELD EMISSION GUN
