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). The 3′ splice site is physically separate from the branch site residue and is not involved in the chemistry of the first-step reaction. 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. 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 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. 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 size distributions of introns and the adjacent exons are similar for both classes of introns. Many genes contain both types of introns in an interspersed pattern, requiring cooperation between the two splicing systems to properly identify the exons. The minor or U12-dependent class has been recognized only relatively recently. The major class, termed here the U2-dependent class, has been the subject of a great deal of investigation for many years. Two types of spliceosomal introns are known to exist in higher eukaryotic plants and animals (see reference 6 for a recent review). Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3′ splice site. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3′ splice site but that formation of the prespliceosomal complex A requires an active 3′ splice site. A strong preference for a spacing of about 12 nucleotides was observed. A branch site-to-3′ splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3′ splice sites. The penultimate A residue, by contrast, was essential for 3′ splice site function. Using an intron with a 5′ splice site AU dinucleotide, any nucleotide could serve as the 3′-terminal nucleotide, although a C residue was most active, while a U residue was least active. U12-dependent introns containing alterations of the 3′ splice site AC dinucleotide or alterations in the spacing between the branch site and the 3′ splice site were examined for their effects on splice site selection in vivo and in vitro.
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