Specialized translational machinery in the Drosophila male germline

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The long-term goal of my research program is to understand post-transcriptional regulation of male germ cell development. Initiation of mRNA translation relies on a 5' cap binding protein called eIF4E, which associates with other translation initiation factors to recruit the ribosome. Metazoan species have evolved multiple eIF4E variants whose in vivo functions are not well defined. For example, mutations in two of the three mouse eIF4E paralogs are associated with distinct neurological defects, and three of the five nematode eIF4E paralogs have unique roles related to fertility. However, it is unclear how the different functions of these eIF4Es are specified. Our data suggest that fruit flies provide an ideal system to address this question. Drosophila melanogaster has eight eIF4E paralogs, of which five are highly expressed in the male germline. We previously showed that eIF4E-3 is needed for successful completion of meiosis (Hernandez et al., 2012). In research funded by NSERC, we knocked out three of the four remaining testis-enriched eIF4Es and discovered that eIF4E-5 is needed at the end of sperm development to separate the mature sperm (Shao et al., in prep.). In male germ cells, the timing and location of eIF4E-3 and eIF4E-5 expression are similar. We therefore hypothesize that the distinct functions of eIF4E-3 and eIF4E-5 are due to differences in their protein sequences and molecular associations. We are therefore uniquely positioned to define the features that enable two eIF4E proteins to play different roles in the same cells. Thus, our specific aims are: Aim 1. Define protein sequences that are important for eIF4E-3 vs. eIF4E-5 function. Aim 2. Identify interacting proteins that are needed for eIF4E-3 vs. eIF4E-5 function. To address these aims, we will perform domain-swapping experiments to define the sequences in eIF4E-3 and -5 that are important for their unique functions in sperm development, and we will use affinity-purification coupled with mass spectrometry (AP-MS) to identify eIF4E-3 and -5 associated proteins. We will validate identified interactions biochemically and use a combination of molecular genetics and phenotypic analysis to test the importance of these interactions in vivo. Our results will define the mechanisms by which these variant Drosophila eIF4Es promote formation of fertile sperm. More broadly, our studies will provide a model for understanding how translation initiation is controlled during development to produce distinct subsets of proteins important for carrying out unique cellular and subcellular functions.