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 of 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 in the splicing of this intron, and a copia long terminal repeat insertion prevents splicing in Drosophila, but not human, extracts. However, a consensus branchpoint does not restore the splicing of introns containing the copia long terminal repeat, and alteration of the wild-type branchpoint sequence alone does not eliminate splicing. These results demonstrate species specificity of splicing signals, particularly pyrimidine stretch and size requirements, and raise the possibility that variant mechanisms not found in mammals may operate in the splicing of small introns in Drosophila and possibly other species.

Purifying selection against spurious splicing signals contributes to the base composition evolution of the polypyrimidine tract

“…Similarly, the position of the branch point relative to the 3′ end, and thus the length of the 3PT, is conserved (Ruskin et al, 1985 ). In contrast to the 5′ side, not only the length, but also the base composition of the 3PT seem important, as some mutations in the polypyrimidine tract and the decrease in its length reduce splicing efficiency (Coolidge et al, 1997 ; Ruskin & Green, 1985 ), while increasing the number of consecutive pyrimidines enhances the removal of the intron (Coolidge et al, 1997 ; Guo et al, 1993 ). Several trans‐splicing factors preferentially bind to the 3PT by identifying motifs, including the essential pre‐mRNA splicing factor U2AF65 which is a part of the U2AF heterodimer (Zamore et al, 1992 ).…”

Purifying selection against spurious splicing signals contributes to the base composition evolution of the polypyrimidine tract

“…Indeed, it serves as the binding site for the required splicing factors (Spellman et al, 2005) and mutations in it result in a decreased splicing efficiency. Increasing pyrimidine content just before the 3’ splice site of a short intron enhanced the removal of the intron, while insertions between the 5’ splice site and the branch point had the opposite effect (Guo et al, 1993). Although the 3PT’s role in splicing is well established, it has not been characterized how its nucleotide composition evolved.…”

“…Several splicing factors preferentially bind to the 3PT. In contrast to the 5’ side, both the size and the base composition seem to be important, as some mutations in the polypyrimidine tract reduce splicing efficiency (Ruskin & Green, 1985; Coolidge et al, 1997), while increasing the pyrimidine content enhances the removal of the intron (Guo et al, 1993).…”

“…Similarly, the position of the branch point relative to the 3’ end, and thus the length of the 3PT, is conserved (Ruskin et al, 1985). In contrast to the 5’ side, not only the size, but also the base composition of the 3PT seem important, as some mutations in the polypyrimidine tract and the decrease in its length reduce splicing efficiency (Ruskin & Green, 1985; Coolidge et al, 1997), while increasing the number of consecutive pyrimidine content enhances the removal of the intron (Guo et al, 1993; Coolidge et al, 1997). Several trans-acting splicing factors preferentially bind to the 3PT by identifying motifs, including the essential pre-mRNA splicing factor U2AF65 which is a part of the U2AF heterodimer (Zamore et al, 1992).…”