The inclusion of exon 16 in the mature protein 4.1R messenger RNA (mRNA) is a critical event in red blood cell membrane biogenesis. It occurs during late erythroid development and results in inclusion of the 10-kd domain needed for stabilization of the spectrin/actin lattice. In this study, an experimental model was established in murine erythroleukemia cells that reproduces the endogenous exon 16 splicing patterns from a transfected minigene. Exon 16 was excluded in predifferentiated and predominantly included after induction. This suggests that the minigene contained exon and abutting intronic sequences sufficient for splicing regulation. A systematic analysis of the cis-acting regulatory sequences that reside within the exon and flanking introns was performed. Results showed that (1) the upstream intron of 4.1R pre-mRNA is required for exon recognition and it displays 2 enhancer elements, a distal element acting in differentiating cells and a proximal constitutive enhancer that resides within the 25 nucleotides preceding the acceptor site; (2) the exon itself contains a strong constitutive splicing silencer; (3) the exon has a weak 5 splice site; and (4) the downstream intron contains at least 2 splicing enhancer elements acting in differentiating cells, a proximal element at the vicinity of the 5 splice site, and a distal element containing 3 copies of the UGCAUG motif. These results suggest that the interplay between negative and positive elements may determine the inclusion or exclusion of exon 16 IntroductionProtein 4.1R is a critical 80-kd cytoskeletal protein found in circulating red blood cells (RBCs). It mediates the formation and maintenance of spectrin/actin complex and anchors the cytoskeleton to the overlying lipid bilayer. 1 Human 4.1R is encoded by a single genomic locus over 200 kb in length, 2 and is expressed as multiple isoforms resulting from complex alternative premessenger RNA (pre-mRNA) splicing pathways. Previous studies have shown that inclusion of a 21-amino acid sequence motif at the N-terminus of the 10-kd spectrin/actin-binding (SAB) domain is required to promote cytoskeletal junctional complex stability. 3,4 Genomic cloning of both the mouse and human genes confirmed that the 63-nucleotide (nt) motif is encoded by an individual exon, exon 16. 2,5 The splicing of this exon is therefore regulated in a cassette fashion. Exon 16 is omitted from much of the 4.1R mRNA of pre-erythroid cells but is included in most of the mRNA produced in late erythroid cells (Figure 1). 6,7 Splicing of exon 16 is thus highly regulated in a differentiation stage-specific manner, and this regulated event is critical for production of 4.1R isoforms that sustain the function of 4.1R in circulating RBCs.Alternative splicing of pre-mRNA is a fundamental mechanism for regulating eukaryotic gene expression. 8 In many cases, alternative RNA splicing contributes to developmentally regulated and cell type-specific patterns of gene expression. Although a great deal of information is available concerning the genera...