An N‐terminal and ankyrin repeat domain interactome of Shank3 identifies the protein complex with the splicing regulator Nono in mice

Abstract

An autism‐associated gene Shank3 encodes multiple splicing isoforms, Shank3a‐f. We have recently reported that Shank3a/b‐knockout mice were more susceptible to kainic acid‐induced seizures than wild‐type mice at 4 weeks of age. Little is known, however, about how the N‐terminal and ankyrin repeat domains (NT‐Ank) of Shank3a/b regulate multiple molecular signals in the developing brain. To explore the functional roles of Shank3a/b, we performed a mass spectrometry‐based proteomic search for proteins interacting with GFP‐tagged NT‐Ank. In this study, NT‐Ank was predicted to form a variety of complexes with a total of 348 proteins, in which RNA‐binding (n = 102), spliceosome (n = 22), and ribosome‐associated molecules (n = 9) were significantly enriched. Among them, an X‐linked intellectual disability‐associated protein, Nono, was identified as a NT‐Ank‐binding protein. Coimmunoprecipitation assays validated the interaction of Shank3 with Nono in the mouse brain. In agreement with these data, the thalamus of Shank3a/b‐knockout mice aberrantly expressed splicing isoforms of autism‐associated genes, Nrxn1 and Eif4G1, before and after seizures with kainic acid treatment. These data indicate that Shank3 interacts with multiple RNA‐binding proteins in the postnatal brain, thereby regulating the homeostatic expression of splicing isoforms for autism‐associated genes after birth.