An ancient competition for the conserved branchpoint sequence influences physiological and evolutionary outcomes in splicing
Branchpoint (bp) recognition is an early event that is required for spliceosome recruitment to a pre-mRNA substrate. The bp sequence can range from nearly invariant in yeast (UACUAAC) to quite degenerate in higher eukaryotes (YUNAY in humans), which correlates with the frequency of alternative splicing observed in the latter. The closely related KH-type RNA binding proteins (RBPs) SF1 and Quaking (QKI) can bind to a similar bp-like sequence, but the extent to which competition for the bp sequence between these two proteins might influence gene expression regulatory mechanisms is unknown. Here we find genomewide evidence for competition between these two RBPs for a subset of alternatively spliced exons that have ACUAAY-like bp sequences. Using a combination of molecular genetic and biochemical techniques with a model intron containing two bp-like sequences, we find that either can be used as a bp but high affinity binding and strong splicing repression by QKI requires both sites. This directly prevents SF1 binding and the subsequent recruitment of spliceosome-associated factors. Finally the ectopic expression of QKI in S. cerevisiae (which lacks QKI) is lethal, due at least in part to widespread splicing repression. In conclusion, QKI can function as a splicing repressor by directly competing with SF1/BBP for a subset of bp sequences that closely mirror its high affinity binding site. This suggests that QKI and degenerate bp sequences may have co-evolved which allowed for expansion of the alternative splicing repertoire in metazoans, plants, and animals. Overall design: We inserted a galactose-inducible cDNA encoding either EGFP, mutant K120A;R124A Qki5, or wildtype Qki5 into BY4741