Alternative splicing in the neural cell adhesion molecule pre-mRNA: Regulation of exon 18 skipping depends on the 5′-splice site

Abstract

Two isoforms of the neural cell adhesion molecule (NCAM), termed NCAM-180 and NCAM-140, derive from a single gene via inclusion or exclusion of the penultimate exon 18 (E18). This alternative splicing event is tissue-specific and regulated during differentiation. To explore its structural basis, we have analyzed the pattern of spliced mRNA generated from transiently transfected minigenes in N2a neuroblastoma cells, which up-regulate E18 usage during differentiation. We show that a minigene construct containing this exon and portions of the adjacent introns and exons faithfully reproduces the differentiation state-dependent alternative splicing of the endogenous pre-mRNA. By systematic deletion and replacement analysis, we scanned the minigene for the presence of functionally important cis-elements. We identified two sequences that affected differentiation state-dependent regulation. One, the central part of E18, does not seem to contain a specific cis-element essential for proper splice site choice, because extending the deletion restored correctly regulated expression of the splicing products. In contrast, the 5′-splice site is an important element for regulation. Replacing it with a corresponding sequence from the α-globin gene resulted in constitutive use of the optional exon. When placed in the ′-globin gene it did not promote alternative splicing. Instead, we observed a strongly decreased efficiency of splicing of the downstream intron in undifferentiated cells. This block of splicing was partially relieved after differentiation. The results are consistent with a model in which skipping of E18 is controlled in part at the associated 5′-splice site by trans-acting factors that undergo quantitative or qualitative changes during differentiation of N2a cells.