ジャーナル: Nat Commun / 年: 2021 タイトル: Structural basis of intron selection by U2 snRNP in the presence of covalent inhibitors. 著者: Constantin Cretu / Patricia Gee / Xiang Liu / Anant Agrawal / Tuong-Vi Nguyen / Arun K Ghosh / Andrew Cook / Melissa Jurica / Nicholas A Larsen / Vladimir Pena / 要旨: Intron selection during the formation of prespliceosomes is a critical event in pre-mRNA splicing. Chemical modulation of intron selection has emerged as a route for cancer therapy. Splicing ...Intron selection during the formation of prespliceosomes is a critical event in pre-mRNA splicing. Chemical modulation of intron selection has emerged as a route for cancer therapy. Splicing modulators alter the splicing patterns in cells by binding to the U2 snRNP (small nuclear ribonucleoprotein)-a complex chaperoning the selection of branch and 3' splice sites. Here we report crystal structures of the SF3B module of the U2 snRNP in complex with spliceostatin and sudemycin FR901464 analogs, and the cryo-electron microscopy structure of a cross-exon prespliceosome-like complex arrested with spliceostatin A. The structures reveal how modulators inactivate the branch site in a sequence-dependent manner and stall an E-to-A prespliceosome intermediate by covalent coupling to a nucleophilic zinc finger belonging to the SF3B subunit PHF5A. These findings support a mechanism of intron recognition by the U2 snRNP as a toehold-mediated strand invasion and advance an unanticipated drug targeting concept.
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 277.15 K / 装置: FEI VITROBOT MARK IV 詳細: The A3'-SSA complex was frozen in vitreous ice by applying ~2.2 uL crosslinked sample to both sides of a glow-discharged UltrAufoil R1.2/1.3 gold grid. The excess sample was blotted away for ...詳細: The A3'-SSA complex was frozen in vitreous ice by applying ~2.2 uL crosslinked sample to both sides of a glow-discharged UltrAufoil R1.2/1.3 gold grid. The excess sample was blotted away for 2s using a blot force of 7 and the grid was snap frozen in liquid ethane cooled by liquid nitrogen..
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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: GIF Quantum LS / エネルギーフィルター - スリット幅: 20 eV
撮影
フィルム・検出器のモデル: GATAN K3 (6k x 4k) / 撮影したグリッド数: 1 / 実像数: 10494 / 平均露光時間: 2.0 sec. / 平均電子線量: 41.41 e/Å2