Sequence and structure of protein binding sites in RNA impact biomolecular condensates

Biomolecular condensates are central to subcellular compartmentalization and RNA regulation. In the multinucleate fungus Ashbya gossypii, condensates composed of Whi3 protein and CLN3 mRNA help ensure nuclear cycle asynchrony in a shared cytoplasm. Here, we investigated how Whi3 protein binding sites within CLN3 mRNA are specified and influence properties of the condensate. We found that Whi3 binds to varied RNA sequences but prefers the five-nucleotide motif UGCAU, which appears at five locations in the CLN3 transcript. Mutating individual UGCAU motifs altered the saturation concentration (Csat) and dense phase concentration of RNA and Whi3 in cell-free reconstitution experiments. These defects were partially rescued by melting and refolding the mRNA, indicating that RNA structure plays a critical role in distinguishing binding sites and determining condensate properties. Lastly, a subset of mutants showed reduced condensate numbers and dysregulation of the cell cycle in cells. These data reveal that the context of otherwise identical mRNA sequences can differentially affect condensate properties.