Role of the 3' splice site in U12-dependent intron splicing.

Two types of spliceosomal introns are known to exist in higher eukaryotic plants and animals (see reference 6 for a recent review). The major class, termed here the U2-dependent class, has been the subject of a great deal of investigation for many years. The minor or U12-dependent class has been recognized only relatively recently. Many genes contain both types of introns in an interspersed pattern, requiring cooperation between the two splicing systems to properly identify the exons. The size distributions of introns and the adjacent exons are similar for both classes of introns. This suggests that the process of splice site recognition for both classes must contend with similar problems of distinguishing correct splice sites amidst the often many tens or hundreds of thousands of nucleotides in a pre-mRNA. The information needed to specify the sites of splicing of both classes of introns is largely located at or near the 5′ and 3′ splice sites. These regions contain conserved sequences that interact with the splicing machinery to promote the assembly of the spliceosome and to specify and activate the chemical cleavage and ligation reactions which lead to the production of spliced RNA. The 3′ splice site region contains two nucleotides that must be precisely located and activated for the two chemical steps in the splicing reaction: the branch site adenosine, with its associated 2′ hydroxyl group, which is the nucleophile in the first step of splicing, and the 3′ splice site residue, which lies immediately adjacent to the phosphodiester bond that is transesterified in the second step of splicing. The 3′ splice site is physically separate from the branch site residue and is not involved in the chemistry of the first-step reaction. Indeed, several studies have shown that the first splicing step can occur in vitro on RNA molecules in which no active 3′ splice site is present due to either truncation of the substrate RNA (1, 13, 33) or mutation of the 3′ splice site AG (15, 31, 39). In U2-dependent introns, the branch site adenosine and the 3′ splice site are generally separated by 11 to 40 nucleotides, although cases are known where they can be over 100 nucleotides apart (18, 37). Positioned between the branch site and the 3′ splice site is a pyrimidine-rich region that appears to be a major recognition element for the 3′ end of an intron. Over the years, much effort has gone into the analysis of these sequence elements to determine the spliceosomal components with which they interact and the role that each plays in specifying the final site of splicing (See references 24 and 30 for recent reviews). Many studies suggest that 3′ splice site selection is largely governed by a 5′-to-3′ scanning mechanism from an independently recognized branch site. Natural introns use the first AG downstream of the branch site as the 3′ splice site (25, 26). This model is also supported by evidence that 3′ splice sites can be located over 100 nucleotides from the branch site (18, 37). This implies that there is no strict limit on how far a 3′ splice site can be from the branch site. Recent in vitro studies using an artificial two-piece trans-splicing system in which the 3′ splice site is supplied on a separate piece of RNA support a model of strict scanning, with the first AG dinucleotide being selected (1, 2, 8). However, other studies have shown that potential 3′ splice sites can compete with one another, suggesting either that scanning is “leaky” or that distal sites can be more active than proximal sites due to distance or sequence preferences (10, 23, 38). A recent study of splicing in the absence of the second-step factor hSlu7 showed that 3′ splice sites both upstream and downstream of the normal 3′ splice site could be used in vitro in the second-step reaction (10). These authors suggest that the normal 3′ splice site is selected but its use is suppressed in the absence of hSlu7. A concomitant effect is that the 5′ exon is more loosely bound to the spliceosome, allowing it to react with normally cryptic 3′ splice sites. The many different systems and organisms used in these studies over the years make it difficult to establish a single mechanistic picture of 3′ splice site selection. It may be that more than one mechanism can be used and the rate-determining step may differ for different introns and for different organisms. For example, similar constructs containing multiple AGs show competition in Saccharomyces cerevisiae (23) but not in mammalian in vitro systems (8). At one extreme are AG-independent introns (31), in which the first splicing step can occur in the absence of a 3′ splice site. In this type of intron, a simple scanning mechanism may suffice. In AG-dependent introns, the 3′ splice site is required for spliceosome formation and the first step of splicing. Here, the 3′ splice site is recognized at least once early in the splicing pathway and then again for the chemical event of the second step. The precise AGs used for the different recognition events may not be the same (47), and likewise, the specific factors and mechanisms of recognition may also differ. Recent results have shown that the small subunit of U2AF interacts with the 3′ splice site AG (23, 46, 48). The role of the small subunit appears to be more significant when the polypyrimidine tract, which interacts with the large subunit, is short or less pyrimidine rich (46). These also appear to be situations in which introns are AG dependent (31). In such circumstances, a strong preference for a 3′ splice site within a preferred distance of the branch site and adjacent to the polypyrimidine tract might be predicted, and indeed, there is evidence of such a constraint (46). AG-independent introns, on the other hand, may not require the small subunit of U2AF to stabilize binding of the large subunit to the polypyrimidine tract, thus allowing spliceosome formation and the first step of splicing to take place without prior selection of a 3′ splice site. In this circumstance, most readily seen in the trans-splicing assay (8), a simple scanning mechanism might then select the 3′ splice site without a strong distance constraint. A distinctly different sequence arrangement is seen in the minor U12-dependent class of introns. These introns lack a polypyrimidine tract and have a highly conserved branch site consensus sequence that appears to be the major recognition element for the 3′ splice site (6, 16, 43). Another characteristic of this class of introns is that the distance between the branch site and the 3′ splice site is short and relatively consistent. An analysis of the information content of U12-dependent 3′ splice sites showed that the branch site consensus contained too little information to uniquely specify a 3′ splice site (6). To further constrain 3′ splice site selection, it would be useful to be able to use the existence of an appropriate 3′ splice site within a set distance of the branch site as an additional recognition element. This requires, however, that the 3′ splice site be recognized along with the branch site element during spliceosome formation. In a recent study, Frilander and Steitz showed that U12-dependent prespliceosome complex formation required the participation of both the 5′ splice site and the branch site (14). They also showed that an RNA which was truncated between the branch site and the 3′ splice site was still competent to form complexes. One interpretation of this finding is that the 3′ splice site does not play a role in the early steps of U12-dependent splicing. In this report, we investigate the nucleotide requirements for a functional U12-dependent 3′ splice site and the consequences of altering the spacing between the branch site and the 3′ splice site both in vivo and in vitro. Our goal was to determine if this distance is mechanistically constrained to the limited range observed so far in native U12-dependent introns and to determine the consequences on splicing and spliceosome formation of violating these constraints. The results suggest that the recognition of the 3′ splice site in U12-dependent introns differs substantially from the process in U2-dependent introns.