Alternative splicing of protein 4.1R exon 16: ordered excision of flankingintrons ensures proper splice site choice

Alternative splicing plays a major role in regulating tissue-specific expre ssion of cytoskeletal protein 4.1R isoforms. In particular, expression of t he protein's functionally critical spectrin actin binding domain, essential for maintenance of red cell membrane mechanical properties, is governed by a developmentally regulated splicing switch involving alternative exon 16. Using a model 3-exon 4.1R pre-messenger RNA (pre-mRNA), we explored the se quence requirements for excision of the introns flanking exon 16. These stu dies revealed that splicing of this alternative exon occurs preferentially in an ordered fashion. The first step is excision of the downstream intron to join exons 16 and 17, followed by excision of the upstream intron, Const ructs designed to test the converse pathway were spliced less efficiently a nd with less fidelity, in part due to activation of a cryptic 5' splice sit e in exon 16. This downstream-first model for ordered splicing is consisten t with the hypothesis that regulated alternative splicing requires cooperat ion between multiple exonic and/or intronic regulatory elements whose spati al organization is critical for recruitment of appropriate splicing factors . Our results predict that exon 16 splicing is regulated at the first step- excision of the downstream intron-and that cells unable to catalyze this st ep will exhibit exon 16 skipping, In cells that include exon 16, adherence to an ordered pathway is important for efficient and accurate production of mature 4.1R mRNA encoding an intact spectrin-actin binding domain.