Hu Zetao, Jin Zhaohui, Guo Lulu, Zeng Ling, Dong Xuanjia, Jiang Lin, Dai Wenting, Ma Jinbiao, Huang Ying
State Key Laboratory of Genetics and Development of Complex Phenotypes, Department of Biochemistry and Biophysics, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200438, China.
Shanghai Key Laboratory of Biliary Tract Disease Research, Department of General Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China.
Nucleic Acids Res. 2025 May 22;53(10). doi: 10.1093/nar/gkaf470.
Ro60 is a conserved RNA-binding protein essential for RNA quality control and implicated in autoimmune responses. In this study, we present the structural and functional characterization of φRo60, an Ro60 homolog from Thermus phage phiLo, with its crystal structure determined at 1.99 Å. Despite limited sequence identity with bacterial and amphibian homologs, φRo60 maintains the canonical doughnut-shaped architecture comprising HEAT repeats and a von Willebrand factor A domain. Surface electrostatic analysis reveals an extensive positively charged region across multiple α-helices, likely facilitating diverse RNA interactions. Moreover, φRo60 binds two Y RNA-like molecules (Yrl1 and Yrl2), identified from the phiLo genome, with distinct stoichiometries, leading to the formation of higher-order nucleocytoplasmic ribonucleoprotein (RNP) complexes. Cryo-electron microscopy of φRo60-Yrl2 RNP complexes revealed a minor population adopting a dimeric assembly, and key positively charged residues are important for φRo60-Yrl2 interactions. Additionally, φRo60 and Yrls interact with host Thermus thermophilus HB8 polynucleotide phosphorylase (PNPase), forming tripartite RYPER-like complexes and attenuating the ribonuclease activity of PNPase. These findings highlight φRo60 as a structurally adaptable Ro60 homolog capable of diverse RNA interactions and host factor recruitment, implying unique strategies for phages to counteract host defense systems in thermophilic environments.
Ro60是一种保守的RNA结合蛋白，对RNA质量控制至关重要，并与自身免疫反应有关。在本研究中，我们展示了来自嗜热栖热菌噬菌体phiLo的Ro60同源物φRo60的结构和功能特征，其晶体结构在1.99 Å分辨率下得以确定。尽管与细菌和两栖动物的同源物序列同一性有限，但φRo60保持了由HEAT重复序列和血管性血友病因子A结构域组成的典型甜甜圈形状结构。表面静电分析揭示了多个α螺旋上广泛的带正电区域，可能有助于与多种RNA相互作用。此外，φRo60与从phiLo基因组中鉴定出的两个Y RNA样分子（Yrl1和Yrl2）以不同的化学计量比结合，导致形成高阶核质核糖核蛋白（RNP）复合物。φRo60 - Yrl2 RNP复合物的冷冻电子显微镜显示，有一小部分采用二聚体组装，关键的带正电残基对φRo60 - Yrl2相互作用很重要。此外，φRo60和Yrls与宿主嗜热栖热菌HB8多核苷酸磷酸化酶（PNPase）相互作用，形成类似RYPER的三方复合物，并减弱PNPase的核糖核酸酶活性。这些发现突出了φRo60作为一种结构适应性强的Ro60同源物，能够进行多种RNA相互作用并招募宿主因子，这意味着噬菌体在嗜热环境中对抗宿主防御系统的独特策略。
