Fox-2A Modulates Protein 4.1R Exon 16 5' Splice Site Activation.

Abstract 778

Erythroid differentiation-induced Fox-2A is an important regulator for the differentiation-specific exon 16 splicing switch in protein 4.1R. Up-regulation of Fox-2A in late erythroid differentiation is critical for inclusion of exon 16, which encodes a portion of the spectrin/actin binding domain vital for maintaining the mechanical stability of red blood cell membranes. Fox-2A exerts its splicing enhancing activity in a motif-dependent manner, binding to UGCAUGs located downstream of exon 16. In this study, we investigated the mechanism by which Fox-2A modulates the expression of exon 16. The excision of introns and the joining of exons depends on the recognition and usage of 5' and 3' splice sites (5' ss and 3' ss, respectively) by the splicing machinery. Exon 16 possesses a relatively strong 3' ss but a weak 5' ss. Mutation of the weak 5' ss (GAGIGTTTGT) to a strong consensus 5' ss (GAGIGTAAGT) led to nearly total exon 16 inclusion. While mutations impairing Fox-2A binding drastically reduced exon 16 inclusion in the presence of the weak 5' ss, no effect on exon 16 inclusion was observed when the strong 5' ss was presented with these mutations. These results suggest that Fox-2A facilitates exon 16 splicing by supporting the weak 5' ss. Early recognition of the 5' ss involves base-pairing interaction with the 5' end of U1 snRNA and stabilization by U1 snRNP. Psoralen-mediated UV cross-linking assays revealed a reduction in U1 snRNA recruitment to the weak 5' ss when Fox-2A binding sites were impaired, suggesting that binding of Fox-2A could promote recruitment and stabilization of U1 snRNP to the weak 5' ss. In support for a role of Fox-2A in modulating the activation of the weak 5' ss by recruiting U1 snRNP, we demonstrated that Fox-2A directly interacts with U1 specific protein U1C in an RNA-independent manner. The C-terminal domain of Fox-2A is responsible for its association with U1C. These data suggest a novel mode for exon 16 5' ss activation in which the binding of Fox-2A to an intronic splicing enhancer element UGCAUG may stabilize the pre-mRNA-U1 snRNP complex through interactions with U1C. These could then result in spliceosome commitment complex formation and exon 16 inclusion.