Zebrafish dead end possesses ATPase activity that is required for primordial germ cell development
Zebrafish dead end possesses ATPase activity that is required for primordial germ cell development
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DOI：
10.1096/fj.09-148403
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发表时间：
2010-08-01
期刊：
影响因子：
4.8
通讯作者：
Collodi, Paul
中科院分区：
文献类型：
--
作者：
Liu, Weiyi;Collodi, Paul
斑马鱼死端(DND)基因在原始生殖细胞(PGC)中特异表达，是PGC迁移和存活所必需的。先前的研究表明，斑马鱼DND的功能是通过保护Nanos1和TDRD7的3‘UTRs免受miR-430b介导的RNA死烯基化的影响。在这项工作中，我们证明了斑马鱼DND蛋白具有形成PGC所需的依赖于镁的ATPase活性。Michaelis-Menten分析表明，在最适条件下，该ATPase的k(CAT)为0.632+/-0.036/m in，利用dnD截短进行的定位研究表明，该酶位于dnD C末端的最后91个氨基酸。该区域的内部缺失和点突变分析用于确定ATPase活性所需的关键氨基酸。通过将编码DND ATPase突变体的mRNAs注射到内源性DND表达被抑制的胚胎中进行的救援实验表明，ATPase活性是斑马鱼PGC正常生存所必需的。实时荧光定量PCR分析表明，当DND缺失ATPase的突变体蛋白在回收的胚胎中表达时，PGC标记Nanos1和TDRD7的表达下调，但不下调VasA的表达，这表明DND ATPase参与了保护Nanos1和TDRD7转录本的过程。FASE B J.24,2641-2650(2010)。Www.fasebj.org
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 Mg2+-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.-Liu, W., Collodi, P. Zebrafish dead end possesses ATPase activity that is required for primordial germ cell development. FASEB J. 24, 2641-2650 (2010). www.fasebj.org