Nuclear 2′- <i>O</i> -methylation regulates RNA splicing through its binding protein FUBP1

2'-<i>O</i>-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 used RNA affinity purification, followed by mass spectrometry to identify N_m-binding proteins. The N_m-binding protein candidates exhibit enriched binding at known N_m sites. Some candidates display nuclear localization and functions. We focused on the splicing factor FUBP1. Electrophoretic mobility shift assay 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 skipped exons, 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, <i>FUBP1</i> 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.