Nuclear 2′-O-methylation regulates RNA splicing through its binding protein FUBP1

2′-O-methylation (N _m ) is an abundant RNA modification exists on different mammalian RNA species. However, potential N _m recognition by proteins has not been extensively explored. Here, we employed RNA affinity purification followed by mass spectrometry to identify N _m -binding proteins. The candidates exhibit enriched binding at known N _m sites. Interestingly, some candidates display nuclear localization and functions. We focused on the splicing factor FUBP1. Electrophoretic mobility shift assay (EMSA) validated preference of FUBP1 to N _m -modified RNA. As FUBP1 predominantly binds intronic regions, we profiled N _m sites in chromatin-associated RNA (caRNA) and found N _m enrichment within introns. Depletion of N _m led to increased exon skipping, suggesting N _m -dependent splicing regulation. The caRNA N _m sites overlap with FUBP1 binding sites, and N _m depletion reduced FUBP1 occupancy on modified regions. Furthermore, FUBP1 depletion induced exon skipping in N _m -modified genes, supporting its role in mediating N _m -dependent splicing regulation. Overall, our findings identify FUBP1 as an N _m -binding protein and uncover previously unrecognized nuclear functions for RNA N _m modification.