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_mrecognition 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_msites. 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_msites in chromatin-associated RNA (caRNA) and found N_menrichment within introns. Depletion of N_mled to increased exon skipping, suggesting N_m-dependent splicing regulation. The caRNA N_msites overlap with FUBP1 binding sites, and N_mdepletion 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_mmodification.