A common mechanism of the regulation of gene expression in metazoans is the alternative splicing of pre-mRNA (6
). Through alternative splicing, multiple mRNAs are generated from the same primary RNA transcript. Changes in splice site choice are regulated by proteins that bind to the pre-mRNA and affect spliceosome assembly. One well-studied family of splicing regulatory factors is the SR proteins that function in both constitutive and alternative splicing and can act at various stages of spliceosome assembly (3
). These proteins are perhaps best known for binding exonic splicing enhancer (ESE) elements and thus stimulating spliceosome assembly at the adjacent splice sites. Some exons bind multiple SR proteins, each of which can activate splicing. Other exons are dependent on a single SR protein. Two of the best-characterized SR protein family members that bind ESEs and activate splicing are SF2/ASF and SC35.
Another large group of proteins that bind to nascent pre-mRNAs are the heterogeneous nuclear ribonucleoproteins (hnRNPs) (19
). At least in vitro, spliceosome assembly occurs after hnRNP binding and some hnRNPs are implicated in splicing regulation. Two of these, hnRNP A1 and polypyrimidine tract binding protein (PTB or hnRNP I) bind to exonic splicing silencer or intronic splicing silencer elements and thus repress the splicing of certain alternatively spliced exons (10
). The mechanisms that hnRNP A1 and PTB use to mediate splicing repression are not fully understood. Several models have been proposed (10
). One mechanism involves direct interactions between positive and negative factors at adjacent binding sites (9
). The concentration of the factors and their relative affinities for their binding sites are what determine exon inclusion or skipping. In other systems, the assembly of multimeric A1 or PTB complexes onto sites surrounding an alternatively spliced exon may cause the intervening region of RNA to loop out and thus prevent splice site recognition (2
). Another possible mechanism of repression by hnRNP A1 involves binding to a high-affinity site within an exon and then promoting the assembly of additional hnRNP A1 molecules along the pre-mRNA (57
). This is thought to create a repressed zone of RNA refractory to the binding of the general splicing machinery at the splice sites. SF2/ASF can block this cooperative propagation of hnRNP A1 along the exon, thereby activating splicing. Both SR proteins and hnRNPs vary in concentration between different cell types, and it is thought that this differential expression of hnRNPs and SR proteins affects the alternative splicing of many pre-mRNAs (7
The N1 exon of c-src
provides a model system for the study of neuron-specific splicing regulation. The 18-nucleotide exon, N1, is included in the mRNA in neurons but is skipped in nonneuronal cells (31
). Two intronic sequences are required for regulation of N1 splicing (1
). The 3′ splice site region upstream of the exon is required for repression of N1 in nonneuronal cells. It has been shown previously that PTB binds to CUCUCU elements (CU) within this sequence and is required for splicing repression in vitro (13
). The second intronic sequence, between 17 and 142 nucleotides downstream of N1, acts as a splicing enhancer and is also required for splicing repression (17
). Within this element is a highly conserved region between nucleotides 37 and 70 called the downstream control sequence (DCS). The DCS contains a CU element that is required for splicing repression. Flanking this CU element are the elements GGGGGCUG and UGCAUG, which are needed for enhancer activity. Each of these elements binds to specific RNA binding proteins: hnRNP F and hnRNP H bind to the G-rich element (16
), KH-type splicing-regulatory protein binds to the UGCAUG element (35
), and PTB or its neural homolog, nPTB, binds to the CU element (17
Studies of splicing regulation, including those on src
N1, have generally focused on either intronic elements that bind PTB and other factors or on exonic elements that bind SR proteins, hnRNP A1, and other proteins. However, it is likely that in many systems the exonic and intronic elements are functioning together. Indeed, in addition to the intronic elements, an element within the N1 exon itself also acts as a splicing enhancer element. When this purine-rich sequence from exon N1, GAGGAAGGUG, is placed within a similarly sized test exon of a heterologous minigene, it activates splicing in vivo (42
). Splicing of the test exon was strongest when both intronic and exonic elements were placed together in the construct, suggesting that they normally work in combination (42
). The enhancement of the test exon by these elements varied between the different neuronal and nonneuronal cell lines tested, but they do not appear to be highly neuron specific.
In this study, we further examine the role of the exonic enhancer in the regulation of exon N1 splicing. We identify the proteins SF2/ASF, hnRNP A1, hnRNP A1B, hnRNP H, and hnRNP F as binding to the element in splicing extracts. We show that SF2/ASF and SC35 can activate splicing of c-src pre-mRNA and that SF2/ASF enhances the splicing pathway leading to inclusion of N1. Both hnRNP A1 and PTB can counteract the activity of the SR proteins to repress splicing but apparently do so by different mechanisms.