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评估人类核 RNA 外切体和 RNA 衔接子复合物的解旋酶和转运活性的方法。

Methods to assess helicase and translocation activities of human nuclear RNA exosome and RNA adaptor complexes.

机构信息

Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, United States.

Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, United States; Howard Hughes Medical Institute, New York, NY, United States.

出版信息

Methods Enzymol. 2022;673:453-473. doi: 10.1016/bs.mie.2022.03.060. Epub 2022 Apr 27.

DOI:10.1016/bs.mie.2022.03.060
PMID:35965016
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9382703/
Abstract

The nuclear RNA exosome collaborates with the MTR4 helicase and RNA adaptor complexes to process, surveil, and degrade RNA. Here we outline methods to characterize RNA translocation and strand displacement by exosome-associated helicases and adaptor complexes using fluorescence-based strand displacement assays. The design and preparation of substrates suitable for analysis of helicase and decay activities of reconstituted MTR4-exosome complexes are described. To aid structural and biophysical studies, we present strategies for engineering substrates that can stall helicases during translocation, providing a means to capture snapshots of interactions and molecular steps involved in substrate translocation and delivery to the exosome.

摘要

核 RNA 外切体与 MTR4 解旋酶和 RNA 衔接复合物协同作用，以加工、监测和降解 RNA。在这里，我们概述了使用基于荧光的链置换测定法来表征与外切体相关的解旋酶和衔接复合物的 RNA 易位和链置换的方法。描述了适合分析重组 MTR4-外切体复合物的解旋酶和衰变活性的底物的设计和制备。为了辅助结构和生物物理研究，我们提出了用于工程化底物的策略，这些底物可以在易位过程中使解旋酶失活，从而提供了一种捕获底物易位和递送至外切体过程中涉及的相互作用和分子步骤的快照的方法。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1e80/9382703/d27ea66c45a8/nihms-1802014-f0005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1e80/9382703/7c7d4db7ee22/nihms-1802014-f0001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1e80/9382703/1d48d987ea2f/nihms-1802014-f0002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1e80/9382703/5b4dddda3ba5/nihms-1802014-f0003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1e80/9382703/65cdd0905490/nihms-1802014-f0004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1e80/9382703/d27ea66c45a8/nihms-1802014-f0005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1e80/9382703/7c7d4db7ee22/nihms-1802014-f0001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1e80/9382703/1d48d987ea2f/nihms-1802014-f0002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1e80/9382703/5b4dddda3ba5/nihms-1802014-f0003.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1e80/9382703/65cdd0905490/nihms-1802014-f0004.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1e80/9382703/d27ea66c45a8/nihms-1802014-f0005.jpg

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本文引用的文献

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RNA helicases are hubs that orchestrate exosome-dependent 3'-5' decay.RNA 解旋酶是协调外切体依赖的 3'-5' 降解的枢纽。
Curr Opin Struct Biol. 2021 Apr;67:86-94. doi: 10.1016/j.sbi.2020.09.010. Epub 2020 Nov 2.
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Distinct and evolutionary conserved structural features of the human nuclear exosome complex.人类核小体外切酶复合物的独特且进化保守的结构特征。
Elife. 2018 Jul 26;7:e38686. doi: 10.7554/eLife.38686.
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Helicase-Dependent RNA Decay Illuminated by a Cryo-EM Structure of a Human Nuclear RNA Exosome-MTR4 Complex.冷冻电镜结构解析人核 RNA 外切体-MTR4 复合物阐明解旋酶依赖的 RNA 降解。
Cell. 2018 Jun 14;173(7):1663-1677.e21. doi: 10.1016/j.cell.2018.05.041.
5
Structural basis for MTR4-ZCCHC8 interactions that stimulate the MTR4 helicase in the nuclear exosome-targeting complex.MTR4-ZCCHC8 相互作用的结构基础，刺激核 exosome 靶向复合物中的 MTR4 解旋酶。
Proc Natl Acad Sci U S A. 2018 Jun 12;115(24):E5506-E5515. doi: 10.1073/pnas.1803530115. Epub 2018 May 29.
6
Structure and reconstitution of yeast Mpp6-nuclear exosome complexes reveals that Mpp6 stimulates RNA decay and recruits the Mtr4 helicase.酵母Mpp6-核外切体复合物的结构与重组表明，Mpp6刺激RNA衰变并招募Mtr4解旋酶。
Elife. 2017 Jul 25;6:e29062. doi: 10.7554/eLife.29062.
7
An Mtr4/ZFC3H1 complex facilitates turnover of unstable nuclear RNAs to prevent their cytoplasmic transport and global translational repression.Mtr4/ZFC3H1 复合物促进不稳定核 RNA 的周转，以防止其细胞质运输和全局翻译抑制。
Genes Dev. 2017 Jun 15;31(12):1257-1271. doi: 10.1101/gad.302604.117. Epub 2017 Jul 21.
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Targeting RNA for processing or destruction by the eukaryotic RNA exosome and its cofactors.靶向RNA以便由真核RNA外切体及其辅助因子进行加工或降解。
Genes Dev. 2017 Jan 15;31(2):88-100. doi: 10.1101/gad.294769.116.
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Identification of a Nuclear Exosome Decay Pathway for Processed Transcripts.鉴定核小体降解途径对加工转录本的作用。
Mol Cell. 2016 Nov 3;64(3):520-533. doi: 10.1016/j.molcel.2016.09.025. Epub 2016 Oct 27.
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Nuclear RNA Exosome at 3.1 Å Reveals Substrate Specificities, RNA Paths, and Allosteric Inhibition of Rrp44/Dis3.3.1埃分辨率下的核RNA外切体复合物揭示了Rrp44/Dis3的底物特异性、RNA路径和变构抑制作用。
Mol Cell. 2016 Nov 17;64(4):734-745. doi: 10.1016/j.molcel.2016.09.038. Epub 2016 Nov 3.