Intranuclear paraspeckle-circular RNA TACC3 assembly forms RNA-DNA hybrids to facilitate MASH-related hepatocellular carcinoma growth in an m6A-dependent manner

Background: Metabolic dysfunction-associated steatohepatitis (MASH) is anticipated to become the leading cause of hepatocellular carcinoma (HCC). Accumulating evidence indicates that N6-methyladenosine (m⁶A)-modified circular RNAs (circRNAs) play key roles in tumor malignant progression. However, the precise molecular mechanisms by which circRNAs and their m⁶A modification regulatory networks respond to metabolic reprogramming, such as lipid overload stress, to drive malignant tumor progression in the context of MASH-related HCC remain unclear. This study aimed to investigate the role and regulatory network of m⁶A-modified circRNAs in MASH-related HCC.

Methods: Epitranscriptomic microarray and in situ hybridization assays were used to validate circTACC3 expression in MASH-related HCC specimens. Palmitic acid (PA) and oleic acid (OA) was applied to NAC-organ assembled three-dimensional-organoid and HCC cell lines to imitate pathological lipid overload. The circTACC3-paraspeckle interaction was studied utilizing fluorescence lifetime imaging microscopy-Forster resonance energy transfer. An integrative analysis combining DNA-RNA immunoprecipitation combined with chromatin isolation by RNA purification (DRIP-ChIRP), γH2AX cleavage under target and tagmentation, and high-throughput/resolution chromosome conformation capture Sequencing were used to study chromatin remodeling induced by circTACC3-formed RNA-DNA hybrids (R loops) at DNA double-strand break (DSB) loci during lipid overload.

Results: The most prevalent m⁶A-modified circRNA in MASH-related HCC, circTACC3, had a substantial impact on the intracellular lipid accumulation, growth, and environmental adaptive survival of tumor cells. Under lipid overload conditions, circTACC3 interacted directly with non-POU domain-containing octamer-binding protein (NONO/p54^nrb) to assemble intranuclear paraspeckle. This process was dependent on the m⁶A-modification sites of circTACC3 and facilitated its nuclear retention. Using DRIP-ChIRP-sequencing, we demonstrated that circTACC3-containing paraspeckles were recruited to DSB foci to form R loops (DSB-circTACC3-R loops). We discovered 4 highly enriched motifs of DSB-circTACC3-R loops. DSB-circTACC3-R loops further facilitated the contact and fusion of topologically associated domains (TADs) and selectively activated genes related to the malignant phenotype of MASH-related HCC. Interestingly, circTACC3-R loops exerted positive feedback control over the assembly of circTACC3 paraspeckle and clustering of TADs.

Conclusions: The m⁶A modification-dependent circTACC3-paraspeckle assembly results in the formation of R loops at DSB foci, leading to chromatin remodeling and the activation of genes involved in MASH-related HCC malignant progression. This process identifies potential therapeutic targets.

DNA double‐strand break; N6‐methyladenosine modification; R loop; chromatin remodeling; circular RNA; hepatocellular carcinoma; lipid overload; metabolic dysfunction‐associated steatohepatitis; paraspeckle.