Institute of Structural and Molecular Biology (ISMB), University College London, London WC1E 6AA, UK.
Department of Structural and Chemical Biology, Center for Biological Research, CIB, CSIC, Av. Ramiro de Maeztu 9, 28040 Madrid, Spain.
Cell Rep Methods. 2023 Jun 26;3(6):100508. doi: 10.1016/j.crmeth.2023.100508.
Understanding how the RNA-binding domains of a protein regulator are used to recognize its RNA targets is a key problem in RNA biology, but RNA-binding domains with very low affinity do not perform well in the methods currently available to characterize protein-RNA interactions. Here, we propose to use conservative mutations that enhance the affinity of RNA-binding domains to overcome this limitation. As a proof of principle, we have designed and validated an affinity-enhanced K-homology (KH) domain mutant of the fragile X syndrome protein FMRP, a key regulator of neuronal development, and used this mutant to determine the domain's sequence preference and to explain FMRP recognition of specific RNA motifs in the cell. Our results validate our concept and our nuclear magnetic resonance (NMR)-based workflow. While effective mutant design requires an understanding of the underlying principles of RNA recognition by the relevant domain type, we expect the method will be used effectively in many RNA-binding domains.
理解蛋白质调节剂的 RNA 结合域如何识别其 RNA 靶标是 RNA 生物学中的一个关键问题，但亲和力非常低的 RNA 结合域在目前用于表征蛋白质-RNA 相互作用的方法中表现不佳。在这里，我们建议使用保守突变来增强 RNA 结合域的亲和力，以克服这一限制。作为原理验证，我们设计并验证了脆性 X 综合征蛋白 FMRP 的一个亲和力增强的 K 同源（KH）结构域突变体，FMRP 是神经元发育的关键调节剂，并使用该突变体来确定该结构域的序列偏好，并解释 FMRP 在细胞中对特定 RNA 基序的识别。我们的结果验证了我们的概念和我们基于核磁共振（NMR）的工作流程。虽然有效的突变设计需要对相关结构域类型识别 RNA 的基本原理有深入的了解，但我们期望该方法将在许多 RNA 结合域中得到有效应用。
