Utilizing functional cell-free extracts to dissect ribonucleoprotein complex biology at single-molecule resolution.

IF 6.4 2区 生物学 Q1 CELL BIOLOGY Wiley Interdisciplinary Reviews: RNA Pub Date : 2023-09-01 Epub Date: 2023-04-12 DOI:10.1002/wrna.1787
Elizabeth Duran, Andreas Schmidt, Robb Welty, Ameya P Jalihal, Sethuramasundaram Pitchiaya, Nils G Walter
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Abstract

Cellular machineries that drive and regulate gene expression often rely on the coordinated assembly and interaction of a multitude of proteins and RNA together called ribonucleoprotein complexes (RNPs). As such, it is challenging to fully reconstitute these cellular machines recombinantly and gain mechanistic understanding of how they operate and are regulated within the complex environment that is the cell. One strategy for overcoming this challenge is to perform single molecule fluorescence microscopy studies within crude or recombinantly supplemented cell extracts. This strategy enables elucidation of the interaction and kinetic behavior of specific fluorescently labeled biomolecules within RNPs under conditions that approximate native cellular environments. In this review, we describe single molecule fluorescence microcopy approaches that dissect RNP-driven processes within cellular extracts, highlighting general strategies used in these methods. We further survey biological advances in the areas of pre-mRNA splicing and transcription regulation that have been facilitated through this approach. Finally, we conclude with a summary of practical considerations for the implementation of the featured approaches to facilitate their broader future implementation in dissecting the mechanisms of RNP-driven cellular processes. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.

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利用功能性无细胞提取物以单分子分辨率剖析核糖核蛋白复合物生物学。
驱动和调节基因表达的细胞机制通常依赖于大量蛋白质和RNA的协调组装和相互作用,这些蛋白质和RNA被称为核糖核蛋白复合物(RNPs)。因此,要完全重组这些细胞机器,并从机制上了解它们是如何在复杂的细胞环境中运作和调节的,这是一项挑战。克服这一挑战的一种策略是在粗的或重组补充的细胞提取物中进行单分子荧光显微镜研究。该策略能够阐明RNP内特定荧光标记生物分子在接近天然细胞环境的条件下的相互作用和动力学行为。在这篇综述中,我们描述了单分子荧光显微镜方法,该方法剖析了细胞提取物中RNP驱动的过程,强调了这些方法中使用的一般策略。我们进一步调查了通过这种方法促进的前信使核糖核酸剪接和转录调控领域的生物学进展。最后,我们总结了实施特色方法的实际考虑因素,以促进其在未来更广泛的实施,剖析RNP驱动的细胞过程的机制。本文分类如下:RNA结构和动力学>RNA结构、动力学和化学RNA与蛋白质和其他分子的相互作用>RNA-蛋白质复合物RNA结构和力学>RNA结构在生物系统中的影响。
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来源期刊
CiteScore
14.80
自引率
4.10%
发文量
67
审稿时长
6-12 weeks
期刊介绍: WIREs RNA aims to provide comprehensive, up-to-date, and coherent coverage of this interesting and growing field, providing a framework for both RNA experts and interdisciplinary researchers to not only gain perspective in areas of RNA biology, but to generate new insights and applications as well. Major topics to be covered are: RNA Structure and Dynamics; RNA Evolution and Genomics; RNA-Based Catalysis; RNA Interactions with Proteins and Other Molecules; Translation; RNA Processing; RNA Export/Localization; RNA Turnover and Surveillance; Regulatory RNAs/RNAi/Riboswitches; RNA in Disease and Development; and RNA Methods.
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