Abstract
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.
Highlights
In eukaryotes, post-transcriptional regulation of gene expression plays important roles in development and differentiation
Immunostaining of ovaries revealed that DmPYM is present in the germarium, nurse cell and follicle cell cytoplasm, and within the oocyte is uniformly distributed in the cytoplasm (Figure S1B)
As oskar mRNA transport to the oocyte posterior pole requires the exon junction complex (EJC) core proteins, and tight control of oskar translation is critical for normal embryonic development [11,12,13,14,15,25], we examined the distribution of oskar mRNA and protein in pym null egg-chambers
Summary
Post-transcriptional regulation of gene expression plays important roles in development and differentiation. These include RNA processing events in the nucleus, such as splicing, which affects 39 end processing of the RNA, mRNA export, localization, translational enhancement and decay [1,2,3,4,5]. In Drosophila, asymmetric localization of several key mRNAs during oogenesis is essential for embryonic patterning [10]. While in transport, these mRNAs are translationally repressed, and protein is produced only upon mRNA localization and at a particular developmental stage. In vivo association of an assembled EJC with oskar has not been shown and the basis for the requirement of the complex in RNA transport remains unclear
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