Novel Exonic Elements That Modulate Splicing of the Human Fibronectin EDA Exon

Abstract

Three exons in the fibronectin primary transcript are alternatively spliced in a tissue-and developmental stage-specific manner. One of these exons, EDA, has been shown previously by others to contain two splicing regulatory elements between 155 and 180 nucleotides downstream of the 3-splice site: an exon splicing enhancer and a negative element. By transient expression of a chimeric ␤-globin/fibronectin EDA intron in COS-7 cells, we have identified two additional exonic splicing regulatory elements. RNA generated by a construct containing the first 120 nucleotides of the fibronectin EDA exon was spliced with an efficiency of approximately 50%. Deletion of most of the fibronectin EDA exon sequences resulted in a 20-fold increase in the amount of spliced RNA, indicative of an exon splicing silencer. Deletion and mutagenesis studies suggest that the fibronectin exon splicing silencer is associated with a conserved RNA secondary structure. In addition, sequences between nucleotides 93 and 118 of the EDA exon contain a non-purine-rich splicing enhancer as demonstrated by its ability to function in a heterologous context.Higher eukaryotes have evolved alternative splicing as a common means of producing several different mRNAs and polypeptides from a single primary transcript (for review, see Ref. 1). Transcripts that undergo alternative splicing can produce mRNAs and proteins with different structures, properties, and functions. The utilization of alternative splice sites can be regulated to prevent the inappropriate accumulation of one or more alternative products. To date, several examples of cisacting regulatory elements, distinct from bona fide splice sites, have been identified which modulate splicing in a diverse assortment of genes from viruses (2-10), Drosophila (11-14), and higher eukaryotes (15-33).The fibronectin gene is a classical example of a gene that undergoes alternative splicing (34, 35). The fibronectin gene is evolutionarily conserved and is predicted to yield up to 20 different mRNAs in human cells. The generation of this remarkable number of different mRNAs, and consequently polypeptides, is made possible by alternative splicing in three different coding regions of the fibronectin primary transcript (for review, see Refs. 36 and 37). One of the alternatively spliced regions encompasses the EDA (also referred to as EIIIA or EDI) exon. This exon is excluded selectively in fibronectin mRNAs produced by hepatocytes, but it is included to various extents by other cell types (Fig. 1A). EDA exon splicing is also subject to developmental regulation as EDA exon inclusion generally declines with age and differentiation (36, 37). In addition to tissue-specific and developmental regulation, inclusion of the EDA exon is modulated during the process of tissue repair and in certain diseases.For these reasons, regulation of fibronectin EDA exon splicing has been the subject of several studies. In fact, the EDA exon was the first exon with which it was demonstrated that proper alternative splicing could occur in...

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