Years of extensive research have provided a detailed understanding of conserved cellular processes and the molecular factors that regulate them. However, as experimental technologies continue to advance, more exceptions to these well-known paradigms have appeared, both in how fundamental cellular processes are regulated and in the diverse functions of individual molecular factors. This thesis examines two such noncanonical phenomena in the model organism Drosophila melanogaster involving two POU/Oct transcription factors. In Papers I and II, we study translational recoding, a process in which the ribosome deviates from the usual rules of mRNA decoding. Specifically, we focus on understanding how the Stop Codon Readthrough (SCR) process is regulated to produce a Drifter protein isoform encoded by drifter/ventral veins lacking (dfr/vvl) in Drosophila. In Paper III, we explore the moonlighting functions of Nubbin (Nub), during mitosis in early Drosophila embryogenesis. 

In Paper I, we identified several cis-acting elements necessary for dfr SCR. Specifically, we found that three mRNA features regulate dfr readthrough: the type of stop codon, the sequence surrounding the translation termination site, and the presence of an mRNA stem-loop. Furthermore, by modifying the sequence and the structure of the dfr stem-loop, we show that its thermodynamic stability strongly influences readthrough efficiency: a more stable structure promotes SCR, whereas a less stable one significantly reduces SCR. Finally, using the structural parameters characterized for the dfr stem-loop, we identified novel stem-loop structures in SCR-candidate genes in Drosophila. 

In Paper II, we identified a miRNA essential for the regulation of dfr SCR in vivo. Using the Gal4-UAS system and cell-culture SCR reporter assays, we show that endogenous levels of this miRNA are critical for dfr SCR, as inhibiting its activity significantly reduces dfr SCR levels. Interestingly, we discovered that this miRNA controls dfr SCR through a conserved target site within the dfr stem-loop. These findings provide insights into the molecular mechanisms governing tissue-specific translational recoding, thereby deepening our understanding of gene expression regulation.

In Paper III, we explored a non-transcriptional role of Nub in early Drosophila embryogenesis. Using advanced genetics and imaging techniques, we discovered that the short isoform, encoded by the nub gene, Nub-PD, is vital for the early, rapid, and synchronous nuclear divisions in Drosophila embryos. In early stages of embryogenesis, Nub-PD specifically localizes around mitotic spindles and is required for spindle assembly, integrity, and dynamics during mitosis. Using a genetic screen, we show that spindle enrichment of Nub-PD depends on motor proteins, mitotic kinases, and a polarity determinant. Interestingly, we observed a similar function for human POU2F1/Oct1 during mitosis, suggesting that POU/Oct factors share equivalent roles in cell division due to their structural and functional similarities.