IscB-ωRNAによるRNA誘導DNA分裂の構造的基礎とCas9とのメカニズム的比較

Gabriel Schuler¹, Chunyi Hu¹, Ailong Ke¹

¹Department of Molecular Biology and Genetics, Cornell University, 253 Biotechnology Building, Ithaca, NY 14853, USA.

Science (New York, N.Y.)

|May 26, 2022

まとめ

Cas9のようなクラス2のCRISPRエフェクターは,トランポゾン核酸から進化した可能性がある. IscB-ωRNA構造はCas9との類似性を明らかにし,DNA分裂メカニズムと ωRNAを説明する.

科学分野:

分子生物学
構造生物学
遺伝学

背景:

クラス2のCRISPR-Casシステムは Cas9とCas12を含む強力な遺伝子編集ツールです
これらのシステムは,IS200/IS605トランポゾンなどの移動性遺伝子要素から進化したと考えられています.
IscBはCas9と同様のドメイン組織を持つ小さなクラス2エフェクタであり,DNAターゲティングと分裂のために ωRNAを使用する.

研究の目的:

二重鎖DNA (dsDNA) 標的に結合したIscB-ωRNA複合体の高解像度構造を解明する.
IscB と Cas9 リボヌクレオプロテインのメカニズムと構造の類似性を明らかにする.
IscB媒介のDNA分裂における ωRNA と特定のドメインの役割を理解する.

主な方法:

IscB-ωRNA-dsDNA複合体の構造を決定するために,冷凍電子顕微鏡 (cryo-EM) が使用された.
分子相互作用を視覚化するために高解像度の構造分析が行われました.
IscB特異的なPLMPドメインの必要性を評価するために,変異分析を使用した.

主要な成果:

2. 78アングストロムの冷凍-EM構造は,IscB-ωRNA-dsDNA複合体内の詳細な相互作用を明らかにした.
ターゲット付近のモチーフ認識,Rループ形成,dsDNA分裂を含む主要なメカニズム的ステップは,高解像度で可視化されました.
ωRNAは,Cas9のRECドメインと類似して機能し,RNA-DNAヘテロデュプレックスと相互作用し,PLMPドメインは,分裂に欠かせないことが判明した.

結論:

この研究は,IscBの構造と機能に関する原子レベルの洞察を提供し,Cas9との進化的関係を強調しています.
この発見は,IscBが,Cas9の機能の側面を反映した,dsDNAの分裂を導くために ωRNAをどのように利用するかを説明する.
祖先のIscBからCas9への移行は, ωRNAの減少とタンパク質領域の進化を含む重要な変化を伴う可能性が高い.

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