RNA functional modulation by Mitoxantrone via RNA structural ensemble repartitioning

Targeting RNA with small molecules offers a strategy to modulate gene expression at undruggable targets. Traditional screens favor thermodynamically stable, low-entropy RNA motifs with defined conformations, yet these provide limited energetic leverage for functional modulation. Many RNAs instead sample dynamic structural ensembles that small molecules can repartition. Using group I self-splicing introns as a model, we identified the antineoplastic drug Mitoxantrone as a competitive inhibitor of RNA self-splicing (IC50 = 4.3 μM) that stabilizes the native conformation of the T4 td intron. Structure-activity analysis showed that the anthraquinone scaffold alone is insufficient, and basic amine-containing side chains are required for RNA structural modulation. Transcriptome-wide chemical probing in human cells revealed preferential binding to GC-rich structured regions, although only a subset showed structural change. Furthermore, global analysis of 5′ UTR ensembles showed altered structural heterogeneity and translation, demonstrating functional repartitioning of RNA conformational landscapes. Mitoxantrone is an FDA-approved anticancer drug that also acts as a general inhibitor of group I intron self-splicing. Here the authors show that Mitoxantrone repartitions RNA conformational ensembles rather than simply binding RNA, stabilizing specific GC-rich structures. This reduces 5′ UTR heterogeneity and increases translation efficiency.