A minor class of pre-mRNA introns whose excision requires a spliceosome containing U11, U12, U4atac/U6atac, and U5 snRNPs has been identified in plants, insects, and vertebrates. We have characterized single loci that specify the U6atac and U12 snRNAs of Drosophila melanogaster. P element-mediated disruptions of the U6atac and U12 genes cause lethality during the third instar larval and embryonic stages, respectively, and are rescued by U6atac and U12 transgenes. The P element disruption of U6atac results in excision defects of U12-type introns from several transcripts including an alternative U12-dependent spliced isoform of prospero, a homeodomain protein required for CNS development. Thus, we demonstrate the requirement for the U12 spliceosome in the development of a metazoan organism.
We have examined the alternative splicing of the Drosophila melanogaster prospero twintron, which contains splice sites for both the U2-and U12-type spliceosome and generates two forms of mRNA, pros-L (U2-type product) and pros-S (U12-type product). We find that twintron splicing is developmentally regulated: pros-L is abundant in early embryogenesis while pros-S displays the opposite pattern. We have established a Kc cell in vitro splicing system that accurately splices a minimal pros substrate containing the twintron and have examined the sequence requirements for pros twintron splicing. Systematic deletion and mutation analysis of intron sequences established that twintron splicing requires a 46-nucleotide purine-rich element located 32 nucleotides downstream of the U2-type 5 splice site. While this element regulates both splicing pathways, its alteration showed the severest effects on the U2-type splicing pathway. Addition of an RNA competitor containing the wild-type purine-rich element to the Kc extract abolished U2-type splicing and slightly repressed U12-type splicing, suggesting that a trans-acting factor(s) binds the enhancer element to stimulate twintron splicing. Thus, we have identified an intron region critical for prospero twintron splicing as a first step towards elucidating the molecular mechanism of splicing regulation involving competition between the two kinds of spliceosomes.Introns are removed from pre-mRNA by two transesterification steps catalyzed by a large multicomponent complex known as the spliceosome. Five small nuclear RNAs, U1, U2, U4/U6, and U5, and more than 60 polypeptides form the active U2-type spliceosome (5). Spliceosome assembly proceeds in an ordered fashion that is directed by the recognition of conserved sequence motifs within the pre-mRNA by small nuclear RNAs and protein factors. These sequences allow efficient and accurate removal of introns and are located at the 5Ј and 3Ј splice sites as well as the branch point (43).A small subset of introns is removed via a unique and divergent spliceosome (U12-type), whose composition and splice site recognition signals differ (22, 56). U11, U12, and U4atac/ U6atac are the functional analogues of U1, U2, and U4/U6 small nuclear RNPs in the U12-type spliceosome; U5 is the only small nuclear RNP shared by both types of spliceosomes (22,32,34,61). As in its U2-type counterpart, interactions of conserved sequence elements at the 5Ј and 3Ј splice sites and branch point with the components of the U12-type spliceosome direct the correct recognition and removal of introns from the pre-mRNA.In addition to the intrinsic quality of the splice sites themselves (19), splice site selection can depend on other properties of the pre-mRNA, such as exon sequences and relative splice site proximity (28, 49), RNA secondary structure (38, 58), exon size (27, 51), and intronic sequences (4). For many premRNAs, the splicing reaction produces only a single product from the pre-mRNA transcript. However, other genes undergo alternative splicing, a process...
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