Single-molecule visualization of sequence-specific RNA binding by a designer PPR protein

Author:

Marzano NicholasORCID,Johnston Brady,Paudel Bishnu P.ORCID,Schmidberger Jason,Jergic SlobodanORCID,Böcking TillORCID,Agostino MarkORCID,Small IanORCID,van Oijen Antoine M.ORCID,Bond Charles S.ORCID

Abstract

AbstractPentatricopeptide repeat (PPR) proteins are a large family of modular RNA-binding proteins that recognize specific ssRNA target sequences. There is significant interest in developing ‘designer’ PPRs for use in diagnostics or as tools to detect and localize target RNA sequences. However, it is unclear how PPRs search for target sequences within complex transcriptomes and current models to predict PPR binding sites struggle to reconcile the effects that RNA mismatches and secondary structure have on PPR binding. To address this, we determined the structure of a designer PPR (dPPR10) bound to its target sequence and used two- and three-colour single-molecule FRET to interrogate the mechanism of ssRNA binding to individual PPR proteins in real time. We demonstrate that longer RNA sequences were significantly slower to bind (or could not bind at all) and that this is, in part, due to their propensity to form stable secondary structures that sequester the target sequence from dPPR10. Importantly, dPPR10 does not associate with non-target flanking sequences, binding specifically to its target sequence within longer ssRNA species. This data provides evidence that PPRs have limited to no capacity to ‘scan’ RNA transcripts for target sequences and instead rely on diffusion for cognate searching. The kinetic constraints imposed by random three-dimensional diffusion may explain the long-standing conundrum of why PPR proteins are abundant in organelles, but almost unknown outside them (i.e. in the cytosol and nucleus). These findings will inform improved prediction of PPR binding sites for the development of designer PPRs.SummaryPentatricopeptide repeat proteins (PPR) are a large family of modular RNA-binding proteins, whereby each module can be ‘designed’ to bind to a specific ssRNA nucleobase and thus any RNA sequence of interest. As such, there is substantial interest in developing ‘designer’ PPRs for a range of biotechnology applications, including diagnostics orin vivolocalisation of RNA species; however, the mechanistic details regarding how PPRs search for and bind to target sequences is unclear. As such, we combined structure-based and single- molecule approaches and determined that PPRs bind only to their target sequences (i.e., they do not associate with non-target RNA sequences) and do not ‘scan’ longer RNA oligonucleotides for the target sequence. Instead, target searching appears kinetically-constrained by random three-dimensional diffusion, providing an explanation as to why PPRs are found almost exclusively in organelle compartments that typically have smaller transcriptomes. Collectively, this work identifies several key considerations for future ‘designer’ PPR developments.

Publisher

Cold Spring Harbor Laboratory

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3