The Rbfox family of splicing factors regulate alternative splicing during animal development and in disease, impacting thousands of exons in the maturing brain, heart and muscle. Rbfox proteins have long been known to bind to the RNA sequence GCAUG with high affinity and specificity, but just half of Rbfox binding sites contain a GCAUG motif in vivo. We incubated recombinant RBFOX2 with over 60,000 mouse and human transcriptomic sequences to reveal substantial binding to several moderate-affinity, non-GCAYG sites at a physiologically relevant range of RBFOX2 concentrations. We find that these ‘secondary motifs’ bind Rbfox robustly in cells and that several together can exert regulation comparable to GCAUG in a trichromatic splicing reporter assay. Furthermore, secondary motifs regulate RNA splicing in neuronal development and in neuronal subtypes where cellular Rbfox concentrations are highest, enabling a second wave of splicing changes as Rbfox levels increase.

Rbfox 剪接因子家族在动物发育和疾病期间调节选择性剪接，影响成熟大脑、心脏和肌肉中的数千个外显子。早就知道 Rbfox 蛋白以高亲和力和特异性与 RNA 序列 GCAUG 结合，但只有一半的 Rbfox 结合位点在体内含有 GCAUG 基序。我们将重组 RBFOX2 与超过 60,000 个小鼠和人类转录组序列一起孵育，以揭示在生理相关的 RBFOX2 浓度范围内与几个中等亲和力的非 GCAYG 位点的实质性结合。我们发现这些“次要基序”在细胞中牢固地结合 Rbfox，并且在三色剪接报告基因检测中，这些基序结合在一起可以发挥与 GCAUG 相当的调节作用。此外，