Stability of orb mRNA in Drosophila ovaries is regulated by cis-regulatory elements in the three prime untranslated region and influences germ cell specification

Orb is one of two Drosophila mRNA-binding CPEB proteins that activate or repress translation by mediating polyadenylation and deadenylation of target mRNAs through interaction with CPE motifs in their 3UTRs. Orb is one of the key regulators of oocyte specification and polarity. This regulation acts through establishment and maintenance of a 3UTR-dependent positive autoregulatory loop. In 16-cell cysts, positive autoregulation is necessary to accumulate high levels of orb mRNA and protein in one of the two pro-oocytes. A previous study showed that deletion of the orb 3UTR fragment containing CPE sites leads to a failure in proper orb mRNA and protein localization; as a result, oocytes are not specified in 16-cell cysts, and egg chambers contain only nurse cells. In the present study, we found that deletion of CPE and CPE-like motifs disrupts proper Orb protein localization and oocyte specification in germaria. CPE motifs are indispensable for orb mRNA stability: deletion of CPE and CPE-like sequences reduces orb mRNA stability to one third of level in wild type. When the 3UTR is deleted, the remaining 115-nucleotide sequence, which does not include CPE motifs, is sufficient for nurse cell development. Removal of this sequence leads to a further decrease in orb mRNA stability and a drop of Orb protein to undetectable levels. Oogenesis stops in region 2a/2b of the germarium, where the checkpoint is localized, and egg chambers do not form. We propose that CPE motifs are required for orb 3UTR-mediated Orb accumulation which drives oocyte specification, whereas low levels of unlocalized Orb protein expressed in the absence of Orb autoregulation are sufficient for nurse cell specification.