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

Abstract

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.