Fox-2 splicing factor binds to a conserved intron motif to promote inclusion of protein 4.1R alternative exon 16.
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.
Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Julie L
JL
Ponthier
Christina
C
Schluepen
Weiguo
W
Chen
Robert A
RA
Lersch
Sherry L
SL
Gee
Victor C
VC
Hou
Annie J
AJ
Lo
Sarah A
SA
Short
Joel A
JA
Chasis
John C
JC
Winkelmann
John G
JG
Conboy
D020816:Amino Acid Motifs; D001483:Base Sequence; D001798:Blood Proteins / Q000378:metabolism / Q000502:physiology*; D002454:Cell Differentiation; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015536:Down-Regulation; D004900:Erythroblasts / Q000378:metabolism; D005091:Exons; D005786:Gene Expression Regulation; D006367:HeLa Cells; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000378:metabolism; D006801:Humans; D007438:Introns; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D000072260:RNA Splicing Factors; D034741:RNA, Small Interfering / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; C455479:RBFOX1 protein, human; C454924:RBFOX2 protein, human; D000072260:RNA Splicing Factors; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; C107467:SF1 protein, human; D014157:Transcription Factors; C028841:erythrocyte membrane band 4.1 protein