In this study, we sought to investigate the mechanism by which heterogeneous nuclear ribonucleoprotein (hnRNP) H and F regulate proteolipid protein (PLP)/DM20 alternative splicing. G-rich sequences in exon 3B, G1 and M2, are required for hnRNPH- and F-mediated regulation of the PLP/DM20 ratio and, when placed between competing 5′ splice sites in an α-globin minigene, direct hnRNPH/F-regulated alternative splicing. In contrast, the activity of the intronic splicing enhancer, which is necessary for PLP splicing, is only modestly reduced by removal of hnRNPH/F both in PLP and α-globin gene context. In vivo, hnRNPH reversed reduction of DM20 splicing induced by hnRNPH/F removal, whereas hnRNPF had little effect. Tethering of the MS2-hnRNPH fusion protein downstream of the DM20 5′ splice site increased DM20 splicing, whereas MS2-hnRNPF did not. Binding of U1 small nuclear ribonucleoprotein (U1snRNP) to DM20 is greatly impaired by mutation of G1 and M2 and depletion of hnRNPH and F. Reconstitution of hnRNPH/F-depleted extracts with either hnRNPH or F restored U1snRNP binding. We conclude that hnRNPH and F regulate DM20 splicing by recruiting U1snRNP and that hnRNPH plays a primary role in DM20 splice site selection in vivo. Decreased expression of hnRNPH/F in differentiated oligodendrocytes may regulate the PLP/DM20 ratio by reducing DM20 5′ splice site recognition by U1snRNP
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