Role of the 3′ splice site consensus sequence in mammalian pre-mRNA splicing

Pre-mRNA splicing has been shown to occur by a two-step pathway1,2. In the first stage, the pre-mRNA is cleaved at the 5′ splice site, generating the first exon RNA species and an RNA species composed of the intron and second exon (IVS1–exon 2 RNA species). In the second stage, cleavage at the 3′ splice site and ligation of the exons occurs, resulting in the excision of the intact intron. The excised intron and IVS1–exon 2 RNA species are in the form of a lariat in which the 5′ end of the intron is joined to an adenosine residue near the 3′ end of the intron by a 2′–5′ phosphodiester bond1–6. Here we show that although cleavage at the 3′ splice site does not occur until the second stage of the splicing reaction, at least a portion of the 3′ splice site consensus sequence is necessary for 5′ splice site cleavage and lariat formation. Thus, in higher eukaryotes at least three sequence elements participate in the initiation of the splicing reaction: the 5′ splice site, 3′ splice site consensus sequence and the RNA branchpoint,