Uncovering essential RNA components of fusion oncoprotein condensates using bicon-seq.

Fusion oncoproteins drive aggressive leukemias by mediating aberrant gene expression within specialized biomolecular condensates that contain various biomolecules. While RNA is known to influence condensate properties, the RNA constituents of fusion protein-containing onco-condensates remain poorly defined. Here, we use bicon-seq (biotinylated-isoxazole-mediated precipitation of condensome and RNA sequencing) to globally profile condensate-associated RNAs from cellular samples. Using experimental perturbation of cytoplasmic RNA-containing condensates, we demonstrated that bicon-seq accurately quantifies changes in RNA solubility, that correlate with published RNA enrichment in these structures. In leukemia cells driven by the NUP98::KDM5A fusion oncoprotein, bicon-seq revealed highly conserved long non-coding RNAs (lncRNAs) that are associated with NUP98::KDM5A onco-condensates. Among them, the lncRNA Gas5 was highly expressed and essential for the proliferation of AML cells. Mechanistically, Gas5 co-localized with NUP98::KDM5A in condensates and modulated NUP98::KDM5A chromatin binding and the expression dynamics of differentiation/apoptosis-associated genes in AML cells. Together, our results introduce a novel approach to systematically profile condensate-associated RNAs and identify the lncRNA Gas5 as a critical regulator of NUP98::KDM5A-driven transcription in AML cells.