Ribonucleoprotein (RNP) granules are membraneless condensates that organize the intracellular space by compartmentalization of specific RNAs and proteins. Studies have shown that RNA tunes the phase behaviour of RNA-binding proteins, but the role of intermolecular RNA–RNA interactions in RNP granules in vivo remains less explored. Here we determine the role of a sequence-specific RNA–RNA kissing-loop interaction in assembly of mesoscale oskar RNP granules in the female Drosophila germline. We show that a two-nucleotide mutation that disrupts kissing-loop-mediated oskar messenger RNA dimerization impairs condensate formation in vitro and oskar granule assembly in the developing oocyte, leading to defective posterior localization of the RNA and abrogation of oskar-associated processing bodies upon nutritional stress. This specific trans RNA–RNA interaction acts synergistically with the scaffold RNA-binding protein, Bruno, in driving condensate assembly. Our study highlights the architectural contribution of an mRNA and its specific secondary structure and tertiary interactions to the formation of an RNP granule that is essential for embryonic development.

核糖核蛋白 （RNP） 颗粒是无膜凝聚物，通过特定 RNA 和蛋白质的区室化来组织细胞内空间。研究表明，RNA 可调节 RNA 结合蛋白的相位行为，但分子间 RNA-RNA 相互作用在体内 RNP 颗粒中的作用仍未得到充分探索。在这里，我们确定了序列特异性 RNA-RNA 吻环相互作用在雌性果蝇种系中尺度 oskar RNP 颗粒组装中的作用。我们表明，破坏接吻环介导的 oskar 信使 RNA 二聚化的双核苷酸突变会损害体外凝聚物的形成和发育卵母细胞中的 oskar 颗粒组装，导致 RNA 的后部定位缺陷和营养应激下 oskar 相关加工体的消除。这种特异性反式 RNA-RNA 相互作用与支架 RNA 结合蛋白 Bruno 协同作用，驱动冷凝物组装。我们的研究强调了 mRNA 的结构贡献及其特定的二级结构和三级相互作用对胚胎发育所必需的 RNP 颗粒的形成。