The 3’UTR of theorb2gene encoding theDrosophilaCPEB translation factor plays a critical role in spermatogenesis

AbstractCPEB proteins are conserved translation regulators involved in multiple biological processes. One of these proteins inDrosophila, Orb2, is a principal player in spermatogenesis. It is required for meiosis and spermatid differentiation. During the later processorb2mRNAs and proteins are localized within the developing spermatid. To evaluate the role oforb2mRNA 3’UTR in spermatogenesis, we used the CRISPR/Cas9 system to generate a deletion of theorb23’UTR,orb2R. This deletion disrupts the process of spermatid differentiation, but has no apparent effect on meiosis. While this deletion appears to destabilize theorb2mRNA and reduce the levels of Orb2 protein, this is not the primary cause of the differentiation defects. Instead, differentiation appears to be disrupted becauseorb2mRNAs and proteins are not properly localized within the differentiating spermatids. Other transcripts and proteins involved in spermatogenesis are also mislocalized inorb2Rspermatids.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 twoDrosophila melanogasterCPEB proteins, is predominantly expressed in the testes and is crucial for spermatogenesis. The 3’UTR oforb2transcript contains multiple CPE-like motifs, which is indicative oforb2self-regulation. We have generated a deletion that removes the greater portion of 3’UTR. While this deletion causes a reduction in the levels oforb2mRNA 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.