The removal of introns from mRNA precursors (pre-mRNAs) involves two relatively straightforward chemical reactions. The recognition of intron-exon boundaries, the splice sites, however, requires the integration of information provided by many cis-acting elements and a complex splicing machinery (64). The cis-acting elements that define the borders between exons and introns are quite diverse and yet are recognized efficiently by the splicing machinery. This machinery is composed of general splicing factors (GSFs), which make up the spliceosome and its associated proteins, and of regulatory factors. The same machinery must also make cell-type-specific choices in cases in which pre-mRNAs are alternatively spliced. This is a monumental task given that it is estimated that transcripts from 30% of all genes in humans are alternatively spliced (http://devnull.lbl.gov:8888/alt).
The spliceosome, like many macromolecular machines, is not preassembled as an active enzyme but rather assembles on the substrate. The substrate, a functional pre-mRNA, is thought to first interact with U1 snRNP, hnRNP proteins, and SR proteins (5, 34). The interaction is determined by RNA-RNA base pairing between the 5′ end of U1 snRNA and the consensus sequence at the 5′ splice site and by interactions mediated by protein factors (34). The protein factors U2AF and SF1 also recognize the polypyrimidine tract, the branch point, and the 3′ splice site, thus bridging the two groups that subsequently will be involved in the first transesterification reaction. This leads to the formation of the commitment complex (CC). The interaction between the protein factors and the 3′ splice site of an internal exon is enhanced by the binding of U1 snRNP at the downstream 5′ splice site (35, 59). This interaction is the basis for exon definition, an idea discussed in greater detail below. The CC and the U2 snRNP interact to yield the prespliceosome, and the branch point sequence is recognized again, albeit differently in this complex. The prespliceosome interacts with a preformed U5-U4-U6 tri-snRNP to form the immature spliceosome, which then undergoes rearrangements that result in the formation of a fully competent enzyme. This interplay of multiple protein factors and RNA components sets the stage for numerous opportunities for and targets of regulation.
The complexity of constitutive and alternative splice site recognition suggests multiple layers of regulation, with each layer the result of combinatorial arrays of elements and factors (38, 48, 64). The first layer is direct sequence recognition that likely occurs early in the formation of the spliceosome. U1 snRNA can read the sequence at the 5′ splice site, and protein factors SF1, U2AF65, and U2AF35 recognize the branch point, the polypyrimidine tract, and the 3′ splice site, respectively (3, 5, 45, 61, 75). These and other GSFs interact with each other and can act as molecular rulers sensing the relative locations of the cis-acting elements. Positional and distance information provides a second layer of discrimination that overlies the detection of individual binding sites. This type of information is transmitted via protein-protein interactions in the definition of exons (2). Another example of this type of distance detection is seen in the α-tropomyosin pre-mRNA (63), where the close proximity of the 5′ splice site of exon 2 to the branch point upstream of exon 3 precludes the inclusion of both exons into the mRNA. Modulation of splice site strength by proteins of the SR family provides yet another layer of regulation (33, 66, 71). SR proteins play roles in constitutive splicing and can be considered GSFs; however, in some instances SR proteins have important roles in alternative splicing. These proteins can be recruited directly to the RNA by enhancer elements in exons or introns or indirectly by interactions with other GSFs (36, 43, 58, 67). hnRNP proteins, some of which bind all pre-mRNAs, can also influence splice site choices, possibly by counteracting SR proteins (6). hnRNP A1 and polypyrimidine tract binding protein (PTB), two proteins classified as hnRNP proteins, repress certain splicing events and thereby provide a layer of negative regulation. Very precise regulation is provided by the existence of cell-type-specific factors; several of these have been described in Drosophila melanogaster (38). The integration of the information in these regulatory layers leads to splice site choice.
Negative regulation of exon inclusion is emerging as a critical layer in splice site choice. Fairbrother and Chasin considered why certain exons are selected, while others, which seem perfectly competent, are ignored (21). These authors suggest that many, and possibly all, exons are under a global repressive influence mediated by many intronic sequences (21). Thus, splice site utilization can be described as a function of both splice site strength and the intensity of the repressive field within a specific region of a pre-mRNA. This global repressive influence can also contribute to the outcome of regulated alternative splicing events, setting the stage for cell-type-specific derepression of exons (1, 8, 12, 46–49, 72, 73). In mammalian cells PTB has been identified as a key splicing repressor. In this review we critically evaluate the role of PTB in exon silencing and speculate on possible mechanisms of its action. We also provide a brief discussion of potential ways in which selective exon inclusion could be achieved by cell-type-specific derepression.