Dynamic regulation of mRNA transport and translation in the Drosophila germline

点击翻译按钮获取中文摘要

mRNA localization is a powerful and evolutionarily conserved strategy that coupled to translational control allows precise spatial and temporal control of gene expression within cells. oskar mRNA, whose protein product induces germline formation and posterior patterning in the Drosophila embryo, is a paradigm for mRNA localization and translational control. Key components regulating oskar ribonucleoprotein particle (RNP) behavior include the Exon Junction Complex (EJC) and the associated spliced oskar localization element (SOLE), the double-stranded RNA-binding proteins Egalitarian and Staufen, and importantly dynein and kinesin-1, microtubule motor proteins of opposite polarity that transport oskar mRNA sequentially from its site of synthesis in the nurse cells to the oocyte posterior pole. To gain deeper insight into RNP assembly and regulation, in the first funding period, we developed a stringent biochemical assay, ipaRT, with which we elucidated the Drosophila EJC binding landscape and the factors affecting EJC positioning transcriptome-wide in adult flies, including on oskar mRNA. We also developed a transcript-specific RNP purification method and applied it to oskar under conditions where the mRNA is either transported or sequestered in processing bodies, and thus identified previously known and new candidate oskar mRNP components, including translation regulators. In the second funding period, we will (1) assess the roles of several of these candidate proteins in RNA regulation in vivo and (2) address the roles of the EJC, Egalitarian and Staufen in mRNP assembly and the switch from dynein to kinesin during oskar mRNA transport.