Identity rather than 3D position informs splicing of rare introns in the human genome.

Three-dimensional genome organization influences splicing, yet how introns, particularly rare introns, are arranged within the nucleus and whether this organization contributes to splicing have not been fully explored. We mapped the organization of six intron classes, major, minor, minor-like, hybrid, major-like, and non-canonical, across four human cell lines using Hi-C, TSA-seq, and DamID-seq. Minor intron-containing genes and their introns were enriched in compartment A and speckle-associated domains (SPADs) and depleted from lamina-associated domains. Integrating TSA-seq with RNA-seq indicated that splicing efficiency depends on intron identity rather than position. For example, despite shared SPAD proximity, major-like and minor-like introns were less efficiently spliced than major and minor introns. While minor introns in SPADs were consistently efficiently spliced, minor introns outside SPADs were more efficiently spliced in cancer cells. These findings reveal subclass-specific intron organization, yet identity rather than position governs splicing efficiency, particularly for minor introns in cancer cells.