Combinatorial Regulation of Protein 4.1R Exon 16 Alternative Splicing: Modulation of Fox-2 Activated Splicing by Other Intronic and Exonic Motifs.

During erythroid differentiation, stage-specific activation of protein 4.1R exon 16 splicing is critical for the mechanical stability of the erythrocyte plasma membrane. The molecular mechanism of this erythroid splicing switch involves multiple factors including stimulation by Fox proteins acting at splicing enhancers in the proximal downstream intron, and inhibition by hnRNPA1 protein acting at silencer elements in exon 16. To explore how the dynamic interplay among these and other factors can fine tune splicing efficiency, we created a series of exon 16-containing minigenes in which splicing efficiency was measured as a function of variation in exon and intron regulatory elements. In the context of wild type exon 16 with intact hnRNP A1 silencer elements and a weak 5′ splice site, an enhancerless construct with no Fox binding sites exhibited little or no exon 16 inclusion in transfected HeLa cells, and over-expression of Fox-2 did not significantly promote inclusion. Insertion of two wild type UGCAUG elements enhanced splicing substantially in a Fox-2-dependent manner and four elements gave even stronger inclusion. Since another study identified the pentamer GCAUG as the Fox binding site, we tested binding site sequence as a potential source of variation in splicing efficiency. Mutation of the first U residue in UGCAUG yielded weaker, but still Fox-2 dependent, activation of splicing, whereas mutation of the terminal G residue dramatically reduced enhancer activity. To investigate whether enhancer activity of Fox binding sites can be modulated by adjacent sequence motifs, we compared exon 16 splicing efficiency in constructs having Fox sites flanked either by neutral sequence or by an A1 silencer element UAGGG. Introduction of the A1 binding site led to a dramatically reduced enhancer activity including its responsiveness to Fox-2 overexpression. These results indicated that efficiency of splicing of Fox-regulated exons is strongly influenced by the number and sequence of intron enhancer elements and by the presence of adjacent antagonistic elements. In further experiments, we demonstrated that the efficiency of splicing is also strongly dependent on exon 16 and its splice sites. Constructs lacking the major exon 16 silencer element for hnRNP A1 binding exhibited partial exon 16 inclusion in the absence, and very strong inclusion in the presence, of a Fox intron enhancer. Finally, strengthening the weak 5′ splice site of exon 16 abrogated many of these regulatory effects and led to strong inclusion of exon 16 independent of other variables. These findings are consistent with previous data showing antagonism between A1 and Fox in their effects on exon 16 splicing, and suggest that Fox proteins primarily function to overcome the weak 5′ splice site and its repression by hnRNP A1 bound at nearby exonic site(s). We propose that the erythroid alternative splicing program can activate splicing a number of alternative exons with variable efficiency based on each exon’s individual complement of exonic and intronic splicing regulatory elements. Modulation of splicing factor expression, typified by the stage-specific down-regulation of hnRNP A1 during erythroblast differentiation, can further alter splicing efficiency of these exons in a selective, motif-dependent manner. Future experiments with exon microarrays will be aimed at identifying some of the alternative exons that are regulated by that program, and determine its importance to the erythroid differentiation process.