Parra, M.K., Gallagher, T.L., Amacher, S.L., Mohandas, N., and Conboy, J.G. (2012) Deep intron elements mediate nested splicing events at consecutive AG-dinucleotides to regulate alternative 3' splice site choice in vertebrate 4.1 genes. Molecular and cellular biology. 32(11):2044-2053.
Distal intraexon (iE) regulatory elements in 4.1R pre-mRNA govern 32 splice site choice at exon 2 (E2) via nested splicing
events, ultimately modulating expression of N-terminal isoforms of cytoskeletal 4.1R protein. Here we explore intrasplicing
in other normal and disease gene context(s) and demonstrate conservation of intrasplicing through vertebrate evolution. In
the paralogous 4.1B gene, we identified <120kb upstream of E2 an ultra-distal intraexon, iEB, that mediates intrasplicing by promoting two intricately-coupled splicing events that ensure selection of a weak distal
acceptor at E2 (E2dis) by prior excision of the competing proximal acceptor (E2prox). Mutating iEB in minigene splicing reporters abrogated intrasplicing, as did blocking endogenous iEB function with antisense morpholinos in live mouse and zebrafish animal models. In a human elliptocytosis patient with a mutant
4.1R gene lacking E2-E4, we showed that aberrant splicing is consistent with iER-mediated intrasplicing at the first available exons downstream of iER, namely alternative E5 and constitutive E6. Finally, analysis of heterologous acceptor contexts revealed a strong preference
for nested 32 splice events at consecutive pairs of AG dinucleotides. Distal regulatory elements may control intrasplicing
at a subset of alternative 32 splice sites in vertebrate pre-mRNAs to generate proteins with functional diversity.