A U1-U3 snRNA-snoRNA interaction couples SF3B1 mutation to chromatin-state rewiring and genome instability.

Mutations in spliceosome factors such as SF3B1 are recurrent across human diseases, including myelodysplastic syndromes and leukemia 1-4 , yet splicing defects alone do not fully explain the widespread chromatin alterations and genome instability in mutant cells 5 . Here, by comprehensively mapping snRNA-directed RNA-RNA interactions, we identify two previously unrecognized interaction motifs in U1 snRNA beyond canonical 5' splice-site pairing 6,7 . These motifs enable U1 RNA to i) bind intronic and other chromatin-associated RNA (caRNA) regions outside of splice sites, and ii) base pair specifically with snoRNA. We uncover a U1-U3 snRNA-snoRNA interaction that recruits the H3K36 methyltransferase SETD2 to caRNA, promoting gene-body H3K36me3 and antagonizing H3K27me3 to modulate chromatin accessibility. The snRNA-snoRNA interface is essential for this previously unrecognized layer of chromatin and transcriptional regulation mediated through SETD2. SF3B1 mutation enhances U1-U3 binding and increases the association of the U1-U3 complex with caRNA, driving chromatin-accessibility remolding, R-loop formation, DNA damage, and copy-number abnormalities that promote tumorigenesis. A U1-specific 2'-O-methoxyethyl antisense oligonucleotide that selectively blocks U1-U3 pairing suppresses these genomic abnormalities, reduces leukemic infiltration, and prolongs survival in xenograft and patient-derived models, establishing pathological snRNA-snoRNA rewiring as a critical driver of SF3B1-mutant leukemogenesis.