Ghosh Sanjay, Obrdlik Ales, Marchand Virginie, Ephrussi Anne
European Molecular Biology Laboratory, Heidelberg, Germany.
PLoS Genet. 2014 Jun 26;10(6):e1004455. doi: 10.1371/journal.pgen.1004455. eCollection 2014 Jun.
In eukaryotes, RNA processing events in the nucleus influence the fate of transcripts in the cytoplasm. The multi-protein exon junction complex (EJC) associates with mRNAs concomitant with splicing in the nucleus and plays important roles in export, translation, surveillance and localization of mRNAs in the cytoplasm. In mammalian cells, the ribosome associated protein PYM (HsPYM) binds the Y14-Mago heterodimer moiety of the EJC core, and disassembles EJCs, presumably during the pioneer round of translation. However, the significance of the association of the EJC with mRNAs in a physiological context has not been tested and the function of PYM in vivo remains unknown. Here we address PYM function in Drosophila, where the EJC core proteins are genetically required for oskar mRNA localization during oogenesis. We provide evidence that the EJC binds oskar mRNA in vivo. Using an in vivo transgenic approach, we show that elevated amounts of the Drosophila PYM (DmPYM) N-terminus during oogenesis cause dissociation of EJCs from oskar RNA, resulting in its mislocalization and consequent female sterility. We find that, in contrast to HsPYM, DmPYM does not interact with the small ribosomal subunit and dismantles EJCs in a translation-independent manner upon over-expression. Biochemical analysis shows that formation of the PYM-Y14-Mago ternary complex is modulated by the PYM C-terminus revealing that DmPYM function is regulated in vivo. Furthermore, we find that whereas under normal conditions DmPYM is dispensable, its loss of function is lethal to flies with reduced y14 or mago gene dosage. Our analysis demonstrates that the amount of DmPYM relative to the EJC proteins is critical for viability and fertility. This, together with the fact that the EJC-disassembly activity of DmPYM is regulated, implicates PYM as an effector of EJC homeostasis in vivo.
在真核生物中，细胞核内的RNA加工事件会影响细胞质中转录本的命运。多蛋白外显子连接复合体（EJC）在细胞核中与剪接过程中的mRNA结合，并在mRNA的输出、翻译、监测和细胞质定位中发挥重要作用。在哺乳动物细胞中，核糖体相关蛋白PYM（HsPYM）结合EJC核心的Y14-Mago异二聚体部分，并可能在翻译的起始轮次中拆解EJC。然而，EJC与mRNA在生理环境中的关联意义尚未得到验证，PYM在体内的功能仍然未知。在这里，我们研究了果蝇中PYM的功能，在果蝇卵子发生过程中，EJC核心蛋白是osk基因mRNA定位所必需的。我们提供证据表明，EJC在体内与osk基因mRNA结合。通过体内转基因方法，我们表明，卵子发生过程中果蝇PYM（DmPYM）N端数量的增加会导致EJC从osk基因RNA上解离，从而导致其定位错误并导致雌性不育。我们发现，与HsPYM不同，DmPYM不与小核糖体亚基相互作用，并且在过表达时以不依赖翻译的方式拆解EJC。生化分析表明，PYM-Y14-Mago三元复合物的形成受PYM C端调节，这表明DmPYM的功能在体内受到调控。此外，我们发现，在正常条件下DmPYM是可有可无的，但其功能丧失对y14或mago基因剂量降低的果蝇是致命的。我们的分析表明，DmPYM相对于EJC蛋白的量对生存力和繁殖力至关重要。这一点，再加上DmPYM的EJC拆解活性受到调控这一事实，表明PYM是体内EJC稳态的效应器。
