Sipl1 and Rbck1 are novel Eya1-binding proteins with a role in craniofacial development

The Eyes absent 1 protein (Eya1) plays an essential role in the development of various organs in both invertebrates and vertebrates. Mutations in the human EYA1 gene are linked to BOR (branchio-oto-renal) syndrome characterized by kidney defects, hearing loss and branchial arch anomalies. For a better understanding of Eya1's function we have set out to identify new Eya1-interacting proteins. Here we report the identification of the related proteins Sipl1 (Shank interacting protein-like 1) and Rbck1 (RBCC protein interacting with PKC 1) as novel interaction partners of Eya1. We confirmed the interactions by GST pull down analysis and co-immunoprecipitation. A first mechanistic insight is provided by the demonstration that Sipl1 and Rbck1 enhance the function of Eya proteins to act as co-activators for the Six transcription factors. Using RT-PCR and in situ hybridization we show that Sipl1 and Rbck1 are co-expressed with Eya1 in several organs during embryogenesis of both mouse and zebrafish. By morpholino-mediated knockdown we demonstrate that the Sipl1 and Rbck1 orthologs are involved in different aspects of zebrafish development. In particular, knockdown of one Sipl1 as well as one Rbck1 ortholog led to a BOR syndrome-like phenotype with characteristic defects in ear and branchial arch formation.