The 3UTR of the orb2 gene encoding the Drosophila CPEB translation factor plays a critical role in spermatogenesis

Abstract

CPEB proteins are conserved translation regulators involved in multiple biological processes. One of these proteins in Drosophila, Orb2, is a principal player in spermatogenesis. It is required for meiosis and spermatid differentiation. During the later process orb2 mRNAs and proteins are localized within the developing spermatid. To evaluate the role of orb2 mRNA 3UTR in spermatogenesis, we used the CRISPR/Cas9 system to generate a deletion of the orb2 3UTR, orb2R. This deletion disrupts the process of spermatid differentiation, but has no apparent effect on meiosis. While this deletion appears to destabilize the orb2 mRNA and reduce the levels of Orb2 protein, this is not the primary cause of the differentiation defects. Instead, differentiation appears to be disrupted because orb2 mRNAs and proteins are not properly localized within the differentiating spermatids. Other transcripts and proteins involved in spermatogenesis are also mislocalized in orb2R spermatids. Author summaryThe conserved family of cytoplasmic polyadenylation element binding (CPEB) proteins can activate or repress translation of target mRNAs, depending on the specific biological context, through interaction with special cytoplasmic polyadenylation element (CPE) sequences. These proteins function mainly in highly polarized cells. Orb2, one of the two Drosophila melanogaster CPEB proteins, is predominantly expressed in the testes and is crucial for spermatogenesis. The 3UTR of orb2 transcript contains multiple CPE-like motifs, which is indicative of orb2 self-regulation. We have generated a deletion that removes the greater portion of 3UTR. While this deletion causes a reduction in the levels of orb2 mRNA and the protein, this does not appear to be responsible for the defects in spermatogenesis observed in the deletion mutant. Instead, it is the mislocalization of the mRNA and protein in the developing spermatids.