Multiple cis elements regulate an alternative splicing event at 4.1R pre-mRNA during erythroid differentiation

The inclusion of exon 16 in the mature protein 4.1 R messenger RNA (mRNA) i s a critical event in red blood cell membrane biogenesis. It occurs during late erythroid development and results in inclusion of the 10-kd domain nee ded for stabilization of the spectrin/actin lattice. In this study, an expe rimental model was established in murine erythroleukemia cells that reprodu ces the endogenous exon 16 splicing patterns from a transfected minigene. E xon 16 was excluded in predifferentiated and predominantly included after i nduction. This suggests that the minigene contained exon and abutting intro nic sequences sufficient for splicing regulation. A systematic analysis of the cls-acting regulatory sequences that reside within the exon and flankin g introns was performed. Results showed that (1) the upstream intron of 4.1 R pre-mRNA is required for exon recognition and it displays 2 enhancer ele ments, a distal element acting in differentiating cells and a proximal cons titutive enhancer that resides within the 25 nucleotides preceding the acce ptor site; (2) the exon itself contains a strong constitutive splicing sile ncer; (3) the exon has a weak 5' splice site; and (4) the downstream intron contains at least 2 splicing enhancer elements acting in differentiating c ells, a proximal element at the vicinity of the 5' splice site, and a dista l element containing 3 copies of the UGCAUG motif. These results suggest th at the interplay between negative and positive elements may determine the i nclusion or exclusion of exon 16. The activation of the enhancer elements i n late erythroid differentiation may play an important role in the retentio n of exon 16. (C) 2001 by The American Society of Hematology.