In mammalian intron splicing, the mechanism by which the 3' splice site AG is accurately and efficiently identified has remained unresolved. We have previously proposed that the 3' splice site in mammalian introns is located by a scanning mechanism for the first AG downstream of the branch point-polypyrimidine tract. We now present experiments that lend further support to this model while identifying conditions under which competition can occur between adjacent AGs. The data show that the 3' splice site is identified as the first AG downstream from the branch point by a mechanism that has all the characteristics expected of a 5'-to-3' scanning process that starts from the branch point rather than the pyrimidine tract. Failure to recognize the proximal AG may arise, however, from extreme proximity to the branch point or sequestration within a hairpin. Once an AG has been encountered, the spliceosome can still see a limited stretch of downstream RNA within which an AG more competitive than the proximal one may be selected. Proximity to the branch point is a major determinant of competition, although steric effects render an AG less competitive in close proximity (-12 nucleotides). In addition, the nucleotide preceding the AG has a striking influence upon competition between closely spaced AGs. The order of competitiveness, CAG = UAG > AAG > GAG, is similar to the nucleotide preference at this position in wild-type 3' splice sites. Thus, 3' splice site selection displays properties of both a scanning process and competition between AGs based on immediate sequence context. This refined scanning model, incorporating elements of competition, is the simplest interpretation that is consistent with all of the available data.Pre-mRNA splicing is a mandatory consequence of the split nature of eukaryotic genes. The production of functional mRNA from primary transcripts requires the accurate removal of the noncoding introns and ligation of adjacent exons. Pre-mRNA splicing provides an important level at which gene expression can be regulated; pairing of different combinations of splice sites leads to the production of tissue-specific mRNAs that code for distinct protein isoforms or that switch off gene expression via the premature termination of open reading frames (52). The splicing pathway proceeds via a well-characterized two-step reaction of successive transesterifications (14,30,39). The integrity of open reading frames demands that the sites of these reactions be specified to a high degree. This specificity is achieved by the presence of consensus sequences at the 5' splice site ([A/C]AG GURAGU) and three elements associated with the 3' end of the intron, the branch point (YNYURAY), the polypyrimidine tract downstream of the branch point, and the 3' splice site (YAGIG) ( (46,48,60,63), while U2 base pairs with the branch point sequence (31,60,61,64). In yeast cells, U5 RNA has been shown to interact with exon sequences adjacent to both splice sites (28) and Ul has recently been shown to base pair with the 3' splice si...

In mammalian intron splicing, the mechanism by which the 3' splice site AG is accurately and efficiently identified has remained unresolved. We have previously proposed that the 3' splice site in mammalian introns is located by a scanning mechanism for the first AG downstream of the branch point-polypyrimidine tract. We now present experiments that lend further support to this model while identifying conditions under which competition can occur between adjacent AGs. The data show that the 3' splice site is identified as the first AG downstream from the branch point by a mechanism that has all the characteristics expected of a 5'-to-3' scanning process that starts from the branch point rather than the pyrimidine tract. Failure to recognize the proximal AG may arise, however, from extreme proximity to the branch point or sequestration within a hairpin. Once an AG has been encountered, the spliceosome can still see a limited stretch of downstream RNA within which an AG more competitive than the proximal one may be selected. Proximity to the branch point is a major determinant of competition, although steric effects render an AG less competitive in close proximity (approximately 12 nucleotides). In addition, the nucleotide preceding the AG has a striking influence upon competition between closely spaced AGs. The order of competitiveness, CAG congruent to UAG > AAG > GAG, is similar to the nucleotide preference at this position in wild-type 3' splice sites. Thus, 3' splice site selection displays properties of both a scanning process and competition between AGs based on immediate sequence context. This refined scanning model, incorporating elements of competition, is the simplest interpretation that is consistent with all of the available data.