RNA G-quadruplex dynamics control gene expression and genome stability through DHX36-mediated regulation

RNA G-quadruplexes (rG4s) serve as critical regulators of gene expression, yet their comprehensive mapping and dynamic regulation in physiological contexts remain technically challenging. Here we develop Ultra-low-input rG4-seq (ULI-rG4-seq), enabling precise rG4 detection in samples as small as 100 cells. This technological advance, combined with acute protein depletion strategies, reveals three fundamental principles of RNA structure regulation: First, we uncover a temporal dimension of rG4 control, where acute and chronic loss of the helicase DHX36 leads to opposing effects on rG4 levels. This observation extends beyond the traditional view of helicases as unwinding enzymes and suggests sophisticated cellular mechanisms maintaining RNA structural homeostasis. Second, through integrated analysis of rG4 landscapes and DHX36 binding profiles, we demonstrate coordination between cytoplasmic rG4 regulation and nuclear gene expression, revealing how RNA structure dynamics orchestrate genome-wide transcriptional programs. This mechanism provides insights into how modulation of a single RNA helicase can broadly impact cellular state. Third, using mouse oocytes as a model system, we establish rG4 regulation as essential for development and female fertility. DHX36 deficiency results in transcriptional defects, genome instability, and premature ovarian failure, demonstrating the physiological importance of proper RNA structure maintenance. These findings advance our understanding of RNA-based regulation and suggest therapeutic strategies for treating disorders of RNA metabolism. The technological and conceptual advances presented here provide new approaches for investigating RNA structure biology in development and disease.