Loss-of-Function Analysis Elucidates Essential Roles of eIF4E Isoforms in Drosophila Spermatogenesis

Background: Through transcriptional and post-transcriptional regulation, eukaryotic cells can control gene expression to moderate vital cell processes and induce morphological changes. In developmental biology, translation initiation is emerging as a key player in gene expression regulation. Translation initiation begins when eukaryotic initiation factor 4E (eIF4E) binds the 5’ mRNA cap to recruit other initiation factors. Eight eIF4E isoforms are present in Drosophila melanogaster. The canonical eIF4E-1 is involved in the translation of all genes and is a common target for translational regulation mechanisms. The activity of testis-specific eIF4Es in Drosophila are largely unclear, but recent evidence on eIF4E-3 suggests that the other isoforms may also possess distinct, essential functions in spermatogenesis. Methods: Here we provide protein localization data and loss-of-function analysis to characterize eIF4E-4, eIF4E-5, and eIF4E-7. Results: Single KD showed few phenotypes, while eIF4E-4/eIF4E-5 double knockdown males had severe de- fects in spermatogenesis. In eIF4E-5/eIF4E-7 double knockdowns, mutations manifested in multiple stages of severity. Conclusions: The unique expression patterns and differential mutant phenotypes observed suggest that the testis-specific isoforms contain varying levels of functional redundancy. eIF4E-4 and eIF4E-5, which share close homology, appear to have overlapping roles in regulating germ cell division during early spermato- genesis. However, during spermatid individualization they seem to assume different functions. eIF4E-7 also appears to be involved in germ cell differentiation, but most likely in a separate mechanism due to the inability of other isoforms to compensate for its knockdown.