Chromatin-associated RNAs (caRNAs) are not only regulatory molecules but also physical scaffolds that may influence 3D genome structure. Although non-coding RNAs like lncRNAs have been linked to chromatin remodeling, the global relationship between caRNAs and chromatin topology had not been fully elucidated. This study aimed to determine how caRNAs influence spatial genome organisation in human cells.
Researchers combined chromatin-associated RNA sequencing (caRNA-seq) with Hi-C to explore caRNA distribution and function in IMR90 cells.
Key technical approaches included:
- Fractionation and sequencing of chromatin-associated RNA (caRNA-seq)
- Genome-wide Hi-C to detect A/B compartments, TADs, and loops
- RNA-seq and chromatin-bound nascent transcript mapping
- CRISPR-mediated depletion of specific caRNAs
- Bioinformatics integration of RNA abundance with chromatin contact maps
- caRNAs preferentially localized to active (A) chromatin compartments, and their abundance strongly correlated with compartment identity.
- Depletion of specific caRNAs (e.g., U2 snRNA) led to compartment switching, revealing caRNAs as active determinants of chromatin state.
- caRNA-rich regions aligned with enhancer-promoter loops, and knockdown disrupted loop strength and contact frequency.
- RNA-binding protein motif analysis revealed a distinct regulatory code for chromatin-interacting RNAs versus cytoplasmic RNAs.
Depletion of chromatin-associated U2 RNA reduces enhancer–promoter loop strength, demonstrating functional impact of caRNAs on 3D genome structure.
This study demonstrated that chromatin-associated RNAs not only mark active genome compartments but also functionally modulate chromatin loops and nuclear architecture. caRNA-seq was instrumental in capturing spatial RNA occupancy and revealing its genome-wide correlation with structural features. These findings support the use of caRNA-seq for dissecting the RNA–chromatin axis in epigenomics and chromatin biology.
GO enrichment analysis of protein-coding genes located near differentially expressed caRNAs. Terms related to stress response, post-translational modification, and GTPase signaling are significantly enriched. 
KEGG pathway enrichment analysis of genes near differentially expressed caRNAs. Enriched pathways include amino acid metabolism, PPAR signalling, and complement cascades. 
Hierarchical clustering heatmap of differentially expressed chromatin-associated RNAs. Red indicates upregulation, blue indicates downregulation. Samples cluster by expression similarity. 
