C-terminal residues specific to Vasa among DEAD-box helicases are required for its functions in piRNA biogenesis and embryonic patterning

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• 作者：Mehrnoush Dehghani ; Paul Lasko
• 关键词：Vasa ; piRNA biogenesis ; Germ plasm ; Embryonic patterning ; Germ cell specification
• 刊名：Development Genes and Evolution
• 出版年：2016
• 出版时间：November 2016
• 年：2016
• 卷：226
• 期：6
• 页码：401-412
• 全文大小：2,064 KB
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Life Sciences
  Developmental Biology
  Neurosciences
  Cell Biology
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-041X
• 卷排序：226

文摘

The DEAD-box RNA helicase Vasa (Vas, also known as DDX4) is required for germ cell development. In Drosophila, analysis of hypomorphic mutations has implicated maternally expressed Vas in germ cell formation and posterior embryonic patterning. vas-null females, which rarely complete oogenesis, exhibit defects in mitotic progression of germline stem cells, Piwi-interacting RNA (piRNA)-mediated transposon silencing, and translation of Gurken (Grk), an EGFR ligand. The carboxy-terminal region of Vas orthologs throughout the animal kingdom consists of several acidic residues as well as an invariant tryptophan in the penultimate or ultimate position (Trp660 in Drosophila melanogaster). Using CRISPR/Cas9 gene editing, we made a substitution mutant in this residue. Replacing Trp660 by Glu (W660E) abolishes the ability of Vas to support germ cell formation and embryonic patterning and greatly reduces Vas activity in piRNA biogenesis, as measured by transposon silencing, and in activating Grk translation. A conservative substitution (W660F) has much milder phenotypic consequences. In addition, females expressing only a form of Vas in which the seven C-terminal amino acids were replaced with the corresponding residues from Belle (Bel, also known as DDX3) show defects in perinuclear nuage assembly and transposon silencing. Oogenesis in females expressing only the chimeric Vas arrests early; however, in a vas1 background, in which early expression of endogenous Vas supports oogenesis, the chimeric protein supports posterior patterning and germ cell specification. These results indicate that the unique C-terminus of Vas is essential for its function in piRNA biogenesis and that the conserved Trp660 residue has an important functional role.