Dissecting RNA selectivity mediated by tandem RNA-binding domains.

IF 4 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Sarah E Harris, Yue Hu, Kaitlin Bridges, Francisco F Cavazos, Justin G Martyr, Bryan B Guzmán, Jernej Murn, Maria M Aleman, Daniel Dominguez
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引用次数: 0

Abstract

RNA-protein interactions are pivotal to proper gene regulation. Many RNA-binding proteins possess multiple RNA-binding domains; however, how these domains interplay to select and regulate RNA targets remains poorly understood. Here, we investigate three multi-domain proteins, Musashi-1, Musashi-2, and unkempt, which share a high degree of RNA specificity, a common feature across RNA-binding proteins. We used massively parallel in vitro assays with unprecedented depth with random or naturally-derived RNA sequences and find that individual domains within a protein can have differing affinities, specificities, and motif spacing preferences. We conducted large scale competition assays between these proteins and determined how individual protein specificities and affinities influence competitive binding. Integration of binding and regulation in cells with in vitro specificities showed that target selection involves a combination of the protein intrinsic specificities described here, but cellular context is critical to drive these proteins to motifs in specific transcript regions. Finally, evolutionarily conserved RNA regions displayed evidence of binding multiple RBPs in cultured cells, and these RNA regions represent the highest affinity targets. This work emphasizes the importance of in vitro and in cultured cells studies to fully profile RNA-binding proteins and highlights the complex modes of RNA-protein interactions and the contributing factors in target selection.

串联RNA结合域介导的解剖RNA选择性。
rna -蛋白相互作用是基因调控的关键。许多rna结合蛋白具有多个rna结合结构域;然而,这些结构域如何相互作用以选择和调节RNA靶标仍然知之甚少。在这里,我们研究了三种多结构域蛋白,Musashi-1, Musashi-2和unkempt,它们具有高度的RNA特异性,这是RNA结合蛋白的共同特征。我们对随机或自然衍生的RNA序列进行了前所未有深度的大量平行体外分析,发现蛋白质中的单个结构域可能具有不同的亲和力、特异性和基序间距偏好。我们在这些蛋白质之间进行了大规模的竞争分析,并确定了单个蛋白质的特异性和亲和力如何影响竞争结合。结合和调控体外特异性的细胞表明,目标选择涉及蛋白质内在特异性的组合,但细胞环境对于驱动这些蛋白质到特定转录区域的基序至关重要。最后,在培养细胞中,进化上保守的RNA区域显示出与多个rbp结合的证据,这些RNA区域代表了最高的亲和力靶点。这项工作强调了在体外和培养细胞中研究rna结合蛋白的重要性,并强调了rna -蛋白相互作用的复杂模式和靶标选择的影响因素。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of Biological Chemistry
Journal of Biological Chemistry Biochemistry, Genetics and Molecular Biology-Biochemistry
自引率
4.20%
发文量
1233
期刊介绍: The Journal of Biological Chemistry welcomes high-quality science that seeks to elucidate the molecular and cellular basis of biological processes. Papers published in JBC can therefore fall under the umbrellas of not only biological chemistry, chemical biology, or biochemistry, but also allied disciplines such as biophysics, systems biology, RNA biology, immunology, microbiology, neurobiology, epigenetics, computational biology, ’omics, and many more. The outcome of our focus on papers that contribute novel and important mechanistic insights, rather than on a particular topic area, is that JBC is truly a melting pot for scientists across disciplines. In addition, JBC welcomes papers that describe methods that will help scientists push their biochemical inquiries forward and resources that will be of use to the research community.
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