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 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, 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.