RNA structure and multiple weak interactions balance the interplay between RNA binding and phase separation of SARS-CoV-2 nucleocapsid.
RNA structure and multiple weak interactions balance the interplay between RNA binding and phase separation of SARS-CoV-2 nucleocapsid.
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DOI：
10.1093/pnasnexus/pgad333
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发表时间：
2023-10
期刊：
影响因子：
--
通讯作者：
Barbar, Elisar J.
中科院分区：
文献类型：
--
作者：
Estelle, Aidan B.;Forsythe, Heather M.;Yu, Zhen;Hughes, Kaitlyn;Lasher, Brittany;Allen, Patrick;Reardon, Patrick N.;Hendrix, David A.;Barbar, Elisar J.
关键词：
SARS-CoV-2的核衣壳（N）蛋白结合病毒RNA，将其浓缩在病毒粒子内，并与RNA相分离以形成液-液浓缩物。对于区分病毒体或液滴中的非序列依赖性N-RNA相互作用与感染细胞内病毒功能所必需的特定基因组RNA（gRNA）基序的N-RNA相互作用的差异，几乎没有共识。为了鉴定RNA结构和负责特异性相互作用和相分离的N结构域，我们使用病毒RNA的前1,000 nt和短RNA片段作为单链和配对RNA的模型。结合亲和力估计从荧光各向异性的这些RNA的两个折叠结构域的N（NTD和CTD）和全长N的比较表明，NTD优先结合单链RNA，虽然它是主要的RNA结合位点，它不是必不可少的相分离。核磁共振光谱鉴定NTD上的两个RNA结合位点：一个先前表征的位点和一个额外的但较弱的RNA结合面，当与主要位点的结合较弱时，例如与dsRNA或结合受损的突变体结合时，该RNA结合面变得突出。具有双链和单链RNA结构的核衣壳结构域的相分离测定支持多个弱相互作用（例如与CTD或NTD的次级面）促进相分离而强的特异性相互作用不促进相分离的模型。这些研究表明，强和多价弱N-RNA相互作用是N.
The nucleocapsid (N) protein of SARS-CoV-2 binds viral RNA, condensing it inside the virion, and phase separating with RNA to form liquid–liquid condensates. There is little consensus on what differentiates sequence-independent N–RNA interactions in the virion or in liquid droplets from those with specific genomic RNA (gRNA) motifs necessary for viral function inside infected cells. To identify the RNA structures and the N domains responsible for specific interactions and phase separation, we use the first 1,000 nt of viral RNA and short RNA segments designed as models for single-stranded and paired RNA. Binding affinities estimated from fluorescence anisotropy of these RNAs to the two-folded domains of N (the NTD and CTD) and comparison to full-length N demonstrate that the NTD binds preferentially to single-stranded RNA, and while it is the primary RNA-binding site, it is not essential to phase separation. Nuclear magnetic resonance spectroscopy identifies two RNA-binding sites on the NTD: a previously characterized site and an additional although weaker RNA-binding face that becomes prominent when binding to the primary site is weak, such as with dsRNA or a binding-impaired mutant. Phase separation assays of nucleocapsid domains with double-stranded and single-stranded RNA structures support a model where multiple weak interactions, such as with the CTD or the NTD's secondary face promote phase separation, while strong, specific interactions do not. These studies indicate that both strong and multivalent weak N–RNA interactions underlie the multifunctional abilities of N.
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影响因子：
2.7
作者：
Helmus JJ;Jaroniec CP
通讯作者：
Jaroniec CP
（1）H，（13）C和（15）N的SARS-COV-2核素蛋白的C末端二聚化结构域的N型骨架化学位移分配。
影响因子：
0.9
作者：
Korn SM;Lambertz R;Fürtig B;Hengesbach M;Löhr F;Richter C;Schwalbe H;Weigand JE;Wöhnert J;Schlundt A
通讯作者：
Schlundt A
DOI：
10.1002/pro.4409
发表时间：
2022-09
期刊：
Protein science : a publication of the Protein Society
影响因子：
--
作者：
通讯作者：
--
DOI：
10.1016/j.jbc.2022.101677
发表时间：
2022-03
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Cascarina SM;Ross ED
通讯作者：
Ross ED
DOI：
10.1002/pro.4221
发表时间：
2022-03
期刊：
Protein science : a publication of the Protein Society
影响因子：
--
作者：
Dang M;Song J
通讯作者：
Song J