Multiple Splicing Defects in an Intronic False Exon

A major question in mammalian pre-mRNA splicing is how exons (or introns) are recognized. Mammalian transcripts are typically tens of thousands of bases long, with large introns alternating with internal exons that are usually less than 300 bases long. How are these small exons recognized within this sea of intronic sequences? Sequences defining a consensus are found at virtually all exon-intron joints (59). At the upstream side of an exon, the 15-nucleotide (nt) 3′ splice site consensus is Y10NCAG/G, and at the downstream side, the 5′ splice site is MAG/GURAGU (M equals A or C; boldface type indicates invariant nucleotides). At a variable distance upstream of the exon (often ∼30 nt) lies the branch point, with the very loose consensus YNYURAY. The AG dinucleotide immediately preceding the exon and the GU dinucleotide immediately following the exon are almost always present. However, considerable variation is found at the other positions: the highest frequency of occurrence of a particular base at a given position ranges from about 35 to 80%. As a result, less than 5% of actual 3′ or 5′ splice sites represent a perfect match to the consensuses described above (our analysis of a database of 2,800 human exons compiled by Reese et al. [http://www.fruitfly.org/sequence/human-datasets.html]). In a typical mammalian transcript, there are many sequences that match these consensuses as well as or better than the sequences at real splice sites yet they are not used for splicing (47, 71). We will refer to these false sites as pseudosites. Real exons are recognized and spliced cotranscriptionally (5, 6, 44, 48, 91) with a half-time of about 5 min in vivo (44); the pseudosites are efficiently ignored during this process. There must be additional signals that distinguish real splice sites from pseudosites or vice versa. These additional recognition elements could act either positively or negatively, and examples of either type have been demonstrated over the last few years. Positive elements, or splicing enhancers, were first recognized as purine-rich sequences that promote splicing when present within some exons (90); more recently, the list of such enhancers has expanded to include AC-rich sequences (24) and intronic locations (13, 38, 58, 76). Despite the demonstration of specific interactions between enhancers and mammalian trans-acting mediators (10, 20, 41, 56, 84), at best only very degenerate motifs have emerged to define the enhancer sequence (51). Enhancers are better understood from the alternative splicing examples for the sex determination genes in drosophila, where several of these sequence elements, along with their trans-acting mediators, having been genetically characterized (for a review, see reference 52). Most enhancers have been identified in the context of alternative splicing; it is not yet clear whether they play an important role in constitutive splicing. The characteristics of the SR family of proteins support this idea, since some of these proteins bind to specific enhancer elements (51, 57, 82) and can also act as essential splicing factors (46, 89). Multiple sequences in constitutively spliced human β-globin exon 2 have been shown to act as enhancers when tested in an alternative splicing framework, and it has been proposed that they function in constitutive exon definition (70). However, their physiological significance within the globin gene is not clear. Our knowledge of the role and mechanism of action of splicing silencers is more limited. It is known that these sequences can act from within an exon (28, 41) or an intron (16, 33, 35, 42), but no consensus sequence for such elements is apparent. In mammals, a silencer-binding protein has been identified in only a few cases (12, 16, 25, 42, 49). A well-studied example in drosophila is the transposase transcript, where the protein PSI has been shown to interact with 5′ pseudosplice sites near the real 5′ splice site to block the alternative splicing of an internal exon (74). Once again, it is not clear that these cases of silencing represent general inhibitory mechanisms operating in constitutive splicing decisions as well. However, the identification of PTB (hnRNP I) (17, 49, 60) and hnRNP A1 (8, 12, 25) as splicing inhibitors raises the possibility of such a role, given the abundance of these proteins as part of hnRNP complexes (30). The effects of premature termination codons on nuclear RNA processing has prompted proposals that the translatability of exons plays a role in their recognition during nuclear RNA processing (15, 88; H. C. Dietz, Letter, Am. J. Hum. Genet. 60:729–730, 1997). In this scenario, the absence of in-frame stop codons in a potential exon could provide the defining criterion for distinguishing real splice sites from pseudosplice sites. However, there is no direct evidence for a cellular mechanism that could capitalize on this information, i.e., recognize the translatability of an exon before it is spliced. Moreover, there are many examples of premature termination codons that do not affect pre-mRNA splicing (e.g., reference 88). A real exon sequence is constrained by its obligation to code for a protein, and so exon sequences do differ statistically significantly from intron sequences, e.g., in the frequency of particular hexanucleotides (94). Recognition of this constraint and a requirement for an open reading frame (ORF) together with the splice consensus sequences allows the prediction of exons within large genes with fairly high reliability (77, 78, 93). However, it is difficult to see how these statistical differences could be exploited to afford molecular recognition. The more likely mechanism is that exon recognition proceeds through the binding of protein or RNA factors to specific sequences or structures. Site-directed mutagenesis of cloned genes and the analysis of mutations in endogenous genes have revealed cis-acting sequences that are necessary for constitutive splicing. In addition to the usual necessity for the conserved GU and AG dinucleotides, these studies have shown absolute or partial dependence on consensus nucleotides in other positions. However, the severity of the phenotype resulting from changes at a given position varies from one splice site to the next (14, 47, 64). In addition, mutations in the exon or the intron outside of the consensus sequence can also have strong effects (14, 64, 67). Some of these sequences have been characterized as enhancers or silencers, as mentioned above, and some of these have been shown to bind specific proteins or snRNPs. In other cases, secondary structure has been shown to play a role in either promoting or inhibiting splicing (3, 32, 34, 40). The general picture that has emerged from these studies is the recognition of the consensus sequences by different snRNPs, with this binding being stabilized either by exon- or intron-bridging interactions with SR and other proteins or by secondary or higher-order structures. However, the specificity and exact nature of these interactions are not clear enough to allow the formulation of rules for exon recognition. Although the requirements for splicing of exons and introns have been extensively investigated, little attention has been paid to the other side of the coin: why are the many sites that match the consensus splice sites, the pseudosites, not used? Do they have perfectly functional splice sites but lack enhancer sequences? Are the false sites defective despite their agreement with the consensus? Are there silencing elements that keep an otherwise functional site from being recognized? We have used a mutational approach to address these questions. Examining the sequence of the large first intron of the human hprt gene, we chose a 3′ pseudosplice site followed by a downstream 5′ pseudosplice site to define a pseudoexon. Rather than using mutation to knock out the splicing function, we asked what it takes to “knock in” splicing, that is, to convert the pseudoexon into a functional exon. We found that despite their apparent similarity to what we understand as splice sites, the intronic pseudosplice sites bordering this pseudoexon were defective. In addition, we found the 3′ pseudosplice site to be associated with an intronic splicing silencer. A requirement for exonic enhancers was not evident in our studies.