Regulation of fibronectin EDA exon alternative splicing: Possible role of RNA secondary structure for enhancer display

The fibronectin primary transcript undergoes alternative splicing in three noncoordinated sites: the cassette-type EDA and EDB exons and the more comp lex IIICS region. We have shown previously that an 81-nucleotide region wit hin the EDA exon is necessary for exon recognition and that this region con tains at least two splicing-regulatory elements: a polypurinic enhancer (ex onic splicing enhancer [ESE]) and a nearby silencer element (exonic splicin g silencer [ESS]). Here, we have analyzed the function of both elements in different cell types. We have mapped the ESS to the nucleotide level, showi ng that a single base change is sufficient to abolish its function. Testing of the ESE and ESS elements in heterologous exons, individually or as part of the complete EDA regulatory region, showed that only the ESE element is active in different contexts. Functional studies coupled to secondary-stru cture enzymatic analysis of the EDA exon sequence variants suggest that the role of the ESS element may be exclusively to ensure the proper RNA confor mation and raise the possibility that the display of the ESE element in a l oop position may represent a significant feature of the exon splicing-regul atory region.