Species-specific signals for the splicing of a short Drosophila intron in vitro.

Abstract

The effects of branchpoint sequence, the pyrimidine stretch, and intron size on the splicing efficiency of the Drosophila white gene second intron were examined in nuclear extracts from Drosophila and human cells. This 74-nucleotide intron is typical of many Drosophila introns in that it lacks a significant pyrimidine stretch and is below the minimum size required for splicing in human nuclear extracts. Alteration of sequences adjacent to the 3' splice site to create a pyrimidine stretch was necessary for splicing in human, but not Drosophila, extracts. Increasing the size of this intron with insertions between the 5' splice site and the branchpoint greatly reduced the efficiency of splicing of introns longer than 79 nucleotides in Drosophila extracts but had an opposite effect in human extracts, in which introns longer than 78 nucleotides were spliced with much greater efficiency. The white-apricot copia insertion is immediately adjacent to the branchpoint normally used splice site is a pyrimidine-rich region. The way in which sequences at the 5' splice site, the branchpoint, the pyrimidine-rich stretch, and the 3' splice site act together in mammalian splicing to specify intron boundaries has been investigated in detail, and much is known of the factors that recognize these sites. For example, the 5' splice site is recognized by the Ul small nuclear ribonucleoprotein (sn-RNP) via base pairing in both mammals and in yeasts (8,34,47,76,78,90), and the branchpoint is similarly recognized by the U2 snRNP (50,58,84,89,91). Binding of the U2 snRNP to the branchpoint requires a number of factors, including the Ul snRNP (2, 67, 75) and U2AF, a factor that binds to the pyrimidine-rich stretch (70).Despite the relatively large amount that is known about the sequence requirements for splicing, it is still not possible to accurately predict the positions of introns from sequence information alone, and the basis of alternative, or regulated, splicing is still being elucidated. The genetics of Drosophila melanogaster has allowed regulatory factors to be identified for several examples of alternative splicing (4,10,11,41,49,85), and it appears that this organism will prove useful for the (25,51,57). In contrast, the branchpoint sequence of mammalian introns has greater flexibility (26,28,50,65,69,88), and the pyrimidine-rich stretch is more important (13,64,68 (5,15,56,60). In We, the level of normally spliced mRNA is greatly reduced, and a number of aberrant RNAs that are polyadenylated within copia are observed (36,46,61,86). Interest in we derives from the existence of mutations in unlinked genes which alter its expression, resulting in increased or decreased eye pigmentation (6,7,11,38,59,62,81,85). Correlation of the structure of a number of derivatives of wa with their phenotypes, the RNAs that they produce, and their response to genetic modifiers has led to the conclusion that there is competition between polyadenylation within copia and the splicing of this intron (31). Furthermore, it appears that the copia inser...

The architecture of pre-mRNAs affects mechanisms of splice-site pairing

“…Using Drosophila nuclear extract (Kc), we were also able to demonstrate additive kinetics for substrates containing the 120-nt intron; however, we were unable to detect sufficient splicing for our substrates containing longer introns (data not shown). These results are consistent with previous in vitro studies demonstrating that splicing of pre-mRNAs with long introns is supported in HeLa nuclear extract but not in Kc extract (15,22,23). The results from all rate experiments are summarized in Fig.…”

“…Lower eukaryotes have a genomic architecture that is typified by small introns and flanking exons with variable length, suggesting that splice-site recognition occurs across the intron (10,13,14). Consistent with this model, expansion of small introns in yeast or Drosophila causes loss of splicing, cryptic splicing, or intron retention (9,15). Taken together, these observations suggest that splice sites are recognized across an optimal nucleotide length.…”

Conservation of the Protein Composition and Electron Microscopy Structure of Drosophila melanogaster and Human Spliceosomal Complexes

“…Furthermore, in contrast to the situation in humans, where more than 90% of introns are longer than 130 nucleotides (nt), a large percentage (54%) of Drosophila introns are less than 80 nt in length, with most having lengths from 59 to 67 nt (29,35). Most of these "short" introns cannot be spliced in mammals, where the minimum intron length required for splicing is ϳ80 nt (18,58). In the case of long introns, splice site pairing is thought to be first established across the exon (so-called exon definition) and, at a later stage, cross-intron interactions are established (4,41).…”

A 31 bp VNTR in the cystathionine β-synthase (CBS) gene is associated with reduced CBS activity and elevated post-load homocysteine levels

“…This hypothesis is supported by studies on Saccharomyces pombe and Drosophila melanogaster that show that expanding small introns in these organisms inhibits splicing of the intron or activates cryptic sites within the expanded introns. 31 The smallest size of intron 13 of the CBS gene is 1196 bp (allele 16) and the longer alleles defined by the 31 bp VNTR lead to relatively large increases in the intron size. The length of intron 13 with 17, 18, 19 and 21 repeat units are respectively, 1227, 1258, 1289 and 1351 bp, adding an additional 2.6, 5.2, 7.8 and 13.0%, respectively.…”