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.分支点（Branchpoint，bp）识别是剪接体招募至前体mRNA底物的必要早期事件。bp序列的保守性在酵母中近乎恒定（为UACUAAC），而在高等真核生物中则高度简并（人类中为YUNAY），这与后者中观察到的可变剪接频率密切相关。亲缘关系密切的KH型RNA结合蛋白（RNA binding proteins，RBPs）SF1与Quaking（QKI）可结合相似的类bp序列，但目前尚不清楚这两种蛋白对bp序列的竞争会在多大程度上影响基因表达调控机制。本研究获得了全基因组水平的证据，证实这两种RBPs会竞争一类携带ACUAAY类bp序列的可变剪接外显子子集。本研究结合分子遗传学与生物化学技术，以包含两条类bp序列的模型内含子为研究对象，发现两条位点均可作为bp发挥功能，但QKI要实现高亲和力结合与强效剪接抑制，则需要同时结合这两个位点。这一过程会直接阻断SF1的结合，并阻碍后续剪接体相关因子的招募。最后，在不含QKI的酿酒酵母（S. cerevisiae）中异位表达QKI会导致细胞致死，这一表型至少部分源于广泛的剪接抑制。综上，QKI可作为剪接抑制因子，通过与SF1/BBP竞争一类与其高亲和力结合位点高度匹配的bp序列子集来发挥功能。这表明QKI与简并bp序列可能存在协同进化，这一过程推动了后生动物、植物与动物体内可变剪接谱的扩张。