Tissue Specific Roles for the Ribosome Biogenesis Factor Wdr43 in Zebrafish Development

Abstract

During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Here we report a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Here we show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate its nucleolar localization and protein interactions in metazoans. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and also ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome.

The Contributions of the Ribosome Biogenesis Protein Utp5/WDR43 to Craniofacial Development

“…The fan mutant embryos exhibited increased apoptosis and reduced proliferation of the developing CNC. Furthermore, fan mutant embryos have decreased steady-state levels of prerRNAs and pre-rRNA processing defects (Zhao et al 2014), as expected from previous studies of the molecular function Utp5/ WDR43 (Gallagher et al 2004;Prieto and McStay 2007). Apoptosis mediated by p53 due to defects in ribosome biogenesis is conserved across species.…”

“…Since depletion or dysfunction of Utp5 or any of its interacting partners in the t-Utp/UTPA subcomplex results in defects in pre-rRNA transcription and processing (Gallagher et al 2004) and because ribosome biogenesis defects result in craniofacial dysmorphology, all the protein-protein interactions of Utp5/WDR43 are likely to have significance in the development of craniofacial structures. This hypothesis is supported by a zebrafish mutant with a premature stop codon in WDR43, which results in a C-terminal truncation that abrogates the interaction between WDR43 and UTP15 and the interaction between WDR43 and UTP4 (Zhao et al 2014). The same mutation in WDR43 that disrupts protein-protein interactions also causes severe craniofacial defects.…”

“…A study that systemically tested whether human proteins could complement their yeast orthologs did not test Utp5/WDR43 (Kachroo et al 2015), although its functional conservation suggests that human WDR43 may complement yeast Utp5. Furthermore, injection of human Wdr43 mRNA partially rescues the zebrafish fantome (fan) / wdr43 mutant craniofacial phenotype (Zhao et al 2014). Overall, there is ample evidence to support a functional conservation for the role of Utp5/WDR43 from yeast to humans.…”

“…Black arrows indicate an interaction found by PCA (Tarassov et al 2008), yeast 2-hybrid (Uetz et al 2000;, in vitro reconstitution (Poll et al 2014) and that the interaction is conserved for the human orthologs of the proteins (Sato et al 2013). Blue crosses indicate protein-protein interactions that are disrupted in fantome mutant zebrafish and result in craniofacial defects (Zhao et al 2014). …”

“…Despite possible alternative functions for these proteins associated with craniofacial defects, all of them are required for efficient transcription of pre-rRNA, thus offering a strong argument that defects in ribosome synthesis-and the likely consequential disruptions in messenger RNA (mRNA) translation and protein synthesis-lie at the heart of the etiology of defective craniofacial development. For this review, we focus our discussion concerning the relationship between ribosome biogenesis and craniofacial development on the protein WDR43, also called Utp5, whose significance in craniofacial development has only recently been identified (Zhao et al 2014). Many erudite reviews have explored the connection between other ribosome biogenesis proteins and craniofacial development (Sakai and Trainor 2009;Trainor and Andrews 2013;van Gijn et al 2013;Ross and Zarbalis 2014;Trainor and Merrill 2014).…”