Formation of polyphasic RNP granules by intrinsically disordered Qβ coat proteins and hairpin-containing RNA

RNA-protein (RNP) granules are fundamental components in mammalian cells where they perform multiple crucial functions. Many RNP granules form via phase separation driven by protein-protein, protein-RNA, and RNA-RNA interactions. Notably, associated proteins frequently contain intrinsically disordered regions (IDRs) which can associate with multiple partners. Previously we have shown that synthetic RNA molecules containing multiple hairpin coat-protein binding sites can phase separate, forming granules capable of selectively incorporating proteins inside. Here, we expand this platform by introducing a phage coat protein with a known IDR which facilitates protein-protein interactions. We show that the coat protein phase-separates on its own in vivo , and that introduction of hairpin-containing RNA molecules can lead to dissolvement of the protein granules. We further demonstrate via multiple assays that RNA valency, determined by the number of hairpins present on the RNA, leads to distinctly different phase behaviors, effectively forming a polyphasic programmable RNP granule. Moreover, by incorporating the gene for a blue fluorescent protein into the RNA, we demonstrate a phase-dependent boost to protein titer. These insights not only shed light on the behavior of natural granules, but also hold profound implications for the biotechnology field, offering a blueprint for engineering cellular compartments with tailored functionalities.