Computational and Functional Analysis of F-box Dependent Ubiquitin Ligases in Xenopus Nervous System Development

Abstract

The development of the central nervous system is a dynamic process during which protein levels are regulated temporally and spatially by synthesis and degradation. While much is known about the regulation of gene expression during development, little is known about the control of protein degradation. Studies of cell cycle regulation show that a major mechanism of protein degradation is through F-box ubiquitin ligases, which function in the recognition and recruitment of specific targets for the Ub/26S proteasome pathway. To understand the role of F-box mediated protein degradation in neurogenesis, we, initially, identified and annotated the F-box family of proteins, with 65 members, in the Xenopus tropicalis genome. Many of these genes are expressed broadly during early Xenopus development and then restricted to the developing organs including the brain, spinal cord, eyes, branchial arches, somites, and heart. To identify the F-box genes that function in neurogenesis, we performed a genome-wide screen in X. tropicalis for targets of REST, the RE-1 silencing transcription factor, which silences neuronal genes in neural progenitors and non-neuronal cells to restrict expression to neurons and identified Fbxo16 ubiquitin ligase. We determined that as expected for a neuronal gene regulated by REST, Fbxo16 is expressed in the differentiating neurons in the brain but excluded from the neural progenitor zone. Loss of function analysis using morpholino knock-down and a dominant negative construct showed that Fbxo16 modulates neuron formation by affecting the function of the proneural protein Neurogenin (Ngn). This is complemented by gain-of-function analysis, which shows elevated neurogenesis with increased Fbxo16. We found that the effect of Fbxo16 on neurogenesis is not through cell cycle regulation but a direct consequence of its ability to regulate proteins required for neurogenesis. In fact, our half-life analyses showed that Fbxo16 stabilizes Ngn, which is a short-lived protein. Our findings suggest that Fbxo16 functions to protect Ngn from degradation to allow its accumulation as neural progenitors differentiate, ensuring the activation of transcriptional targets.