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

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