Anti-RBFOX2 (full length) polyclonal antibody

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 family of proteins that consists of Rbfox1, Rbfox2, and Rbfox3 in mammals regulates alternative pre-mRNA splicing in various tissues via direct binding to their RNA binding element. Although many studies have indicated the splicing activity of each member of the Rbfox family, the interactions of Rbfox family proteins are largely unknown. Here, we have investigated interactions among Rbfox family proteins. Co-immunoprecipitation (Co-IP) and GST-pull down assays confirmed that Rbfox proteins form homo and hetero complexes. Moreover, in vivo crosslinking using disuccinimidyl suberate treatment indicated that the Rbfox proteins form a dimer which then assembles with other proteins to form a large multiprotein complex. Duolink in situ proximity ligation (PLA) assay revealed that neuron specific Rbfox3 protein interacts with other Rbfox family proteins. This study is the first to provide an evidence that Rbfox family proteins form homo- and hetero-oligomeric complexes in vivo. (C) 2017 Elsevier Inc. All rights reserved.