A Novel Splicing Factor RBM-9 Regulates Protein 4.1R Exon 16 Splicing.

Abstract

Protein 4.1R (4.1R), a vital component of the red cell membrane cytoskeleton, stabilizes the spectrin-actin lattice and attaches it to embedded membrane proteins. A regulated splicing event, the inclusion of exon 16 that encodes for peptides critical for spectrin/actin binding, occurs during late erythroid differentiation. We showed earlier that an intricate combination of enhancer and silencer elements direct exon 16 splicing. Regulated expression of splicing factors, SF2/ASF and hnRNP A/B, has also been implicated in mediating exon 16 splicing. In this study, we attempted to characterize the mechanism involved in exon 16 splicing through UGCAUG, an intronic splicing enhancer present in three copies in the intron downstream of exon 16. We first used a wild-type minigene construct consisting of exons 13, 16, 17 and their respective flanking introns that mimics endogenous exon 16 splicing during the induced differentiation of mouse erythroleukemia cells (MELC). Mutational analysis showed a dose-dependent effect of UGCAUG on exon 16 splicing: the presence of all three copies had the most effect. These results were recapitulated with an internal chimeric exon in a test neutral reporter system “DUP4-1”, suggesting that the enhancing effect could be attributed directly to UGCAUG. Furthermore, we identified a novel splicing factor, RBM-9, from MELC that enhanced the internal exon splicing in an UGCAUG-dependent manner in both the exon 16 minigene and DUP4-1 reporter systems. Our characterization of RBM-9 revealed that diverse isoforms of RBM-9 are generated by the utilization of alternative translation initiation sites and tissue-specific alternative splicing; different isoforms from various tissues exhibited differential exon 16 splicing enhancing activities. MELC-RBM-9 enhanced exon 16 splicing the most among all RBM-9 isoforms tested. Inhibition of RBM-9 expression by RBM-9-shRNA reversed its enhancing activity on exon 16 inclusion in MELC. RBM-9-shRNA also reduced exon 16 splicing in a dose-dependent manner in HeLa cells. Furthermore, purified RBM-9 specifically binds to the UGCAUG sequence in a gel-mobility shift assay. Finally, expression of RBM-9 is upregulated and correlates with exon 16 inclusion during MELC differentiation. These results suggest that a novel splicing factor, RBM-9, enhances erythroid differentiation stage-specific exon 16 splicing by interacting with the splicing enhancer UGCAUG.