Heterogeneous nuclear ribonucleoproteins H and F regulate the proteolipid protein/DM20 ratio by recruiting U1 small nuclear ribonucleoprotein through a complex array of G runs.
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 alpha-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 alpha-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.
Wang
Erming
E
Department of Neurology, University of Kentucky, Lexington, Kentucky 40536, USA.
Cambi
Franca
F
D017398:Alternative Splicing; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D018991:Myelin Proteolipid Protein / Q000737:chemistry*; D009836:Oligodendroglia / Q000737:chemistry; D011485:Protein Binding; D011510:Proteolipids / Q000737:chemistry*; D012326:RNA Splicing; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism*; D055542:alpha-Globins / Q000378:metabolism
D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H; D018991:Myelin Proteolipid Protein; C472930:PLP1 protein, human; D011510:Proteolipids; D017412:Ribonucleoprotein, U1 Small Nuclear; D055542:alpha-Globins