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*
- 1 To whom correspondence should be addressed: University of Kentucky, Dept. of Neurology, Kentucky Clinic L445, Lexington, KY 40536. Tel.: 859-323-5683; Fax: 859-323-5943; E-mail: franca.cambi{at}uky.edu.
Abstract
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.
Footnotes
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↵2 The abbreviations used are: U1snRNP, U1 small nuclear ribonucleoprotein; U1snRNA, U1 small nuclear RNA; hnRNP, heterogeneous nuclear ribonucleoprotein; PLP, proteolipid protein; siRNA, small interference RNA; RT, reverse transcription; MS, MS2 coat protein binding motif; NR, non-related sequence; WT, wild type; MT, mutated; Bt2cAMP, dibutyryl cyclic AMP; ISE, intronic splicing enhancer.
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↵* This work was supported by National Institutes of Health Grant RO1NS053905 from NINDS. This work was also supported by the European Leukodystrophy Association. The mass spectrometric analysis was performed at the University of Kentucky, Center for Structural Biology Protein Core Facility. This core facility is supported in part by funds from NCRR/NIH Grant P20 RR020171.
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4 and Table S1.
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- Received December 12, 2008.
- Revision received February 24, 2009.
- The American Society for Biochemistry and Molecular Biology, Inc.
