U2AF binding selects for the high conservation of the C. elegans 3' splice site

Abstract

Caenorhabditis elegans is unusual among animals in having a highly conserved octamer sequence at the 3' splice site: UUUU CAG/R. This sequence can bind to the essential heterodimeric splicing factor U2AF, with U2AF65 contacting the U tract and U2AF35 contacting the splice site itself (AG/R). Here we demonstrate a strong correspondence between binding to U2AF of RNA oligonucleotides with variant octamer sequences and the frequency with which such variations occur in splice sites. C. elegans U2AF has a strong preference for the octamer sequence and exerts much of the pressure for 3' splice sites to have the precise UUUUCAG/R sequence. At two positions the splice site has a very strong preference for U even though alternative bases can also bind tightly to U2AF, suggesting that evolution can select against sequences that may have a relatively modest reduction in binding. Although pyrimidines are frequently present at the first base in the exon, U2AF has a very strong bias against them, arguing there is a mechanism to compensate for weakened U2AF binding at this position. Finally, the C in the consensus sequence must remain adjacent to the AG/R rather than to the stretch of U's, suggesting this C is recognized by U2AF35.

FIGURE 1.

U2AF⁶⁵ and U2AF³⁵ bind preferentially to the 3′ splice site consensus oligonucleotide. (A) Graphic representation of the C. elegans 3′ splice site consensus sequence from >28,000 confirmed introns. The height of each letter represents the frequency of each base found at that position according to the intron profile (Kent and Zahler 2000). Black letters indicate consensus bases. (B) Oligonucleotides with substitutions at positions −3 and +1 were ³²P-labeled, mixed with whole cell extract, ultraviolet-cross-linked, and immunoprecipitated with antibodies to U2AF⁶⁵.

FIGURE 2.

Competition for U2AF⁶⁵ binding to the consensus sequence by oligonucleotides with single nucleotide substitutions. Graphic representation of competition experiments with oligonucleotides containing single nucleotide substitutions at the indicated position. Transgenic extract was ultraviolet cross-linked with ³²P-labeled U₄CAGG-containing oligonucleotides in the presence of variant competitors. Increasing amounts of competitor oligonucleotides were in 2.5-fold, fivefold, 10-fold, and 25-fold molar excess over labeled RNA, which was always present at 8 ng. Bands were quantified and normalized using the PhosphorImager system from Molecular Dynamics. U indicated by red circles; A, green squares; C, blue diamonds; and G, black triangles.

FIGURE 3.

Competition for U2AF⁶⁵ binding to the consensus sequence by oligonucleotides with multiple substitutions. Note the partial suppression of the effect of a −5 A substitution by a concomitant A substitution at −4 but not −6.

FIGURE 4.

Competition for U2AF⁶⁵ binding to the consensus sequence by oligonucleotides with multiple base insertions. In the lower panel, an AA insertion between the −4 and −3 positions has a minimal effect on binding, whereas an AA added between the −3 and −2 positions dramatically reduces binding. In the upper panel, a two-base insertion is tolerated far more than a five-base insertion.

FIGURE 5.

Different modes of recognition of the 3′ splice site in different species. In the yeast, Saccharomyces cerevisiae, introns are AG independent. The splice site is recognized by tight binding of SF1/BBP to the highly conserved branchpoint consensus sequence. In mammals, the site is tripartite; it is identified by weaker binding of SF1/BBP to this site, as well as by binding of the polypyrimidine tract by U2AF⁶⁵ and the YAG by U2AF³⁵. In C. elegans the octamer at the splice site is bound tightly by the U2AF^65/35 complex.