Fox-2 splicing factor binds to a conserved intron motif to promote inclusion of protein 4.1R alternative exon 16

被引:95
作者
Ponthier, JL
Schluepen, C
Chen, WG
Lerscho, RA
Gee, SL
Hou, VC
Lo, AJ
Short, SA
Chasis, JA
Winkelmann, JC
Conboy, JG
机构
[1] Lawrence Berkeley Natl Lab, Life Sci Div, Berkeley, CA 94720 USA
[2] Univ Cincinnati, Coll Med, Div Hematol Oncol, Dept Internal Med,Vontz Ctr Mol Studies, Cincinnati, OH 45267 USA
关键词
D O I
10.1074/jbc.M511556200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of heterogeneous nuclear ribonucleoprotein ( hnRNP) A/B proteins to silencer elements in the exon and that down-regulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This article demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox- 2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2. These findings suggest that Fox- 2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.
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收藏
页码:12468 / 12474
页数:7
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