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MPP6によるヒト核内エキソソーム複合体の活性および基質特異性の制御機構

藤原, 奈央子 京都大学 DOI:10.14989/doctor.r13551

2023.03.23

概要

ゲノム情報の正確な発現において、適切な RNA 分解は不可欠である。RNA
エキソソーム(以後、エキソソーム)は真核生物において主要な 3’-5’エキソリボヌク
レアーゼ活性を担う複合体であり、RNA 代謝の幅広い局面を制御している。エキソソ
ームは、コアと呼ばれるヌクレアーゼ活性を持たないリング状の構造を基本とし、コ
アに付随するヌクレアーゼや補因子を複数種持つことで多様な基質 RNA に対応して
いる。ヒト核内では、RRP6 と DIS3 がヌクレアーゼとしてコアに直接結合する。ま
た、核特異的な補因子である RNA ヘリカーゼ MTR4 は、現在までに同定された複数
のエキソソーム補因子複合体に共通して含まれており、核内でのエキソソームによる
基質分解に必須の因子である。In vitro における複合体の再構成解析から、RRP6 およ
び核特異的な補因子の一つである MPP6 は、それぞれコアと MTR4 に同時に結合す
ることで MTR4-コア間の相互作用に介在し、MTR4 依存性の基質 RNA 分解を活性化
することが示されている。一方、実際の細胞内における MTR4-コア間の相互作用の重
要性についての知見は未だ非常に乏しい。また、補因子 MPP6 の細胞内におけるエキ
ソソーム機能への関与についても、古くにヒトの rRNA プロセシング異常や酵母の遺
伝学的相互作用を指標とした研究で示唆されたのみで、その作用機序や MPP6 が分解
に寄与する基質 RNA の範囲等についての詳細は不明であった。
本論文では、ヒト核内エキソソームによる基質 RNA 分解における MPP6 の
役割の解明を目指し解析を行った。エキソソームに付随するヌクレアーゼや補因子を
単独あるいは同時にノックダウンし、基質 RNA 分解および MTR4-コア間相互作用へ
の影響を観察することで、エキソソーム複合体内における各因子の機能および因子間
の機能的な相互作用を評価した。その結果、MPP6 は RRP6 と DIS3 による MTR4 依
存的な poly(A)+ RNA 基質分解のそれぞれを促進することを明らかにした。また、
MPP6 はヒト核内においても MTR4 とコアの結合に介在し、その結合様式はこれまで
に in vitro 解析で明らかにされたものとよく合致することを明らかにした。一方、
RRP6 による MTR4 のコアへのリクルート量が維持されている状況でも、MTR4 依存
性の poly(A)+ RNA 基質分解は MPP6 ノックダウンで阻害されることから、MPP6 と
RRP6 それぞれが介在する MTR4-コア間相互作用の間に機能的な差異を見出した。こ
れは比較的単純な基質 RNA を扱ってきたこれまでの in vitro 解析では捉えられてこな
かった結論で、ヒト細胞を用いて多様かつ複雑な基質 RNA への影響を評価すること
により初めて見出された成果である。さらに、MPP6 が標的とする基質 RNA の範囲
とその特徴を把握することを目的として、次世代シーケンシング解析を行い、各エキ
ソソーム構成因子ノックダウンに対する感受性に基づいて基質 RNA を分類した。 ...

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