Abstract
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.