Title : circHIPK3 regulates nucleolin phase separation in aging: Insights from AI-driven predictionandexperimental validation
Abstract:
Cardiac aging is closely linked to cardiomyocyte (CM) senescence. Ribosome biogenesis plays acritical role in triggering CM cell cycle reentry; however, the specific activators and upstreamregulatorsofthis process remain unclear. Circular RNAs (circRNAs) have emerged as key regulators in various cardiacphysiological and pathological processes. Among them, circHIPK3 has been implicated in maintainingcardiac homeostasis. Nucleolin (NCL), a known regulator of ribosome biogenesis, is proposed to exert itsfunctions through liquid–liquid phase separation (LLPS), and its activity may be modulated by circHIPK3. In this study, we aimed to investigate whether circHIPK3 mediates its role in cardiac aging throughNCLLLPS. We developed a novel three-stage iterative artificial intelligence (AI) framework to predict theLLPSpotential of NCL. CM-specific circHIPK3 knockout (CKO) mice were generated, with a lifespan not exceeding 18 months. Using animal models along with RNA pull-down, FRAP, and Ribo-Halo assays, weexamined the interaction between NCL and circHIPK3. AI-based analysis revealed that NCL possesses anintrinsic capacity for LLPS, which progressively declines during aging. In murine cardiac tissue, circHIPK3expression decreased with advancing age. Knockout of circHIPK3 led to cardiac dysfunction, myocardial fibrosis, and multiple aging-related phenotypes, including activation of the p21 signaling pathway. At themolecular level, circHIPK3 directly binds to the 5′ untranslated region (5′UTR) of NCL mRNA, therebyenhancing NCL mRNA stability and sustaining NCL-dependent LLPS to promote ribosome biogenesis. Furthermore, overexpression of circHIPK3 in CKO mice effectively restored NCL expression and suppressed the aging-associated p21 pathway. In conclusion, we identified a novel regulatory axis—the circHIPK3–NCL LLPS–ribosome biogenesis pathway—that protects against cardiac aging via post-transcriptional mechanisms. These findingsunderscore the pivotal role of circHIPK3 in maintaining cardiac homeostasis during aging and suggest that circHIPK3 may serve as both a biomarker and a therapeutic target for age-related cardiac dysfunction.
