Zebrafish dead end possesses ATPase activity that is required for primordial germ cell development

Abstract

Zebrafish dead end (dnd) mRNA is specifically expressed in primordial germ cells (PGCs) and is required for PGC migration and survival. Previous studies have shown that zebrafish Dnd functions by protecting the 3'UTRs of nanos1 and TDRD7 from miR-430b-mediated RNA deadenylation. In this work, we demonstrate that zebrafish Dnd protein possesses Mg(2+)-dependent ATPase activity that is required for PGC formation. Michaelis-Menten analysis revealed that the ATPase has a k(cat) of 0.632 +/- 0.036/min under optimal conditions, and mapping studies using Dnd truncates showed that ATPase resides in the last 91 aa of the Dnd C terminus. Internal deletion and point mutagenesis analysis of this region were used to identify key amino acids required for ATPase activity. Rescue experiments conducted by injecting mRNAs encoding the Dnd ATPase mutants into embryos in which the endogenous dnd expression was inhibited demonstrated that the ATPase activity is required for normal zebrafish PGC survival. Real-time PCR analysis showed that the expression of PGC markers nanos1 and TDRD7 but not vasa were down-regulated when dnd mutant proteins lacking ATPase were expressed in the rescued embryos, indicating that the Dnd ATPase is involved in protecting nanos1 and TDRD7 transcripts.