Figure 1. Exon inclusion is influenced by multiple elements and factors. The inclusion or skipping of a typical cassette exon (gray box) is affected by multiple sequence elements, in addition to the consensus splice site elements. ESE = exon splicing enhancers (green); ISE = intron splicing enhancers (blue); ESS = exon splicing enhancers (red); ISS = intron splicing silencers (orange). Typically, ESEs bind SR proteins, while silencers tend to bind hnRNP proteins.

Figure 2. Exon definition. (a) Experimental evidence leading to the exon definition hypothesis [ 26]. Top: A constitutively spliced model three‐exon, two‐intron pre‐mRNA substrate. Middle: Mutation of the 5′SS of the internal exon leads to exon skipping rather than retention of intron 1. Bottom: Expansion of the central exon by insertion of nonspecific spacer sequences led to exon skipping when the size of the exon exceeded 300 nt; (b) Schematic diagram of how the spliceosomal E and A complexes could form as exon definition complexes. The complexes would form via numerous protein–RNA, protein–protein, and RNA–RNA interactions. The SR proteins can contact the exonic RNA via interaction of their RRM domains with an ESE, and make protein–protein interactions with U2AF ³⁵ and U170K via their RS domains. The interaction of the SRp RS domain with the branch point in the A complex is not depicted.

Figure 3. Neuron‐specific alternative splicing of c‐src N1 exon. Upper panel: In non‐neuronal cells (e.g., HeLa), exon N1 is skipped. PTB binds cooperatively to sequences on both sides of the N1 exon. This does not prevent binding of U1 snRNP at the N1 5′SS, but it does prevent productive cross‐intron interactions between the bound U1 snRNP and an exon‐definition complex formed across exon 4. As a result, exon 4 splices to exon 3. Lower panel: In neuronal cells (e.g., WERI), PTB is replaced by the less‐repressive nPTB. An enhancer complex assembles on the downstream control sequence, involving nPTB, the neuronally expressed Fox‐1 and Fox‐2 proteins, and the generally expressed hnRNP‐F, hnRNP‐H, and KSRP. In addition, the SR protein SF2/ASF binds to an ESE. As a result, N1 exon is included between exons 3 and 4 of c‐src mRNA.

Figure 4. Position‐dependent splicing activity. Binding of Fox proteins on the upstream side of cassette exons is associated with the skipping of cassette exons, while binding on the downstream side is associated with exon inclusion.