Unraveling the molecular basis of ao, a maternal-effect gene in Drosophila melanogaster

Abstract

The abnormal oocyte (ao) gene of Drosophila melanogaster is a maternal-effect, embryonic semi-lethal gene located on the left arm of the 2nd chromosome. ao was recovered from a wild D. melanogaster population and initially described by Larry Sandler and colleagues in 1968. Subsequent studies found that increasing heterochromatic regions on the X, Y, and 2nd chromosomes rescued the maternal-effect lethality of ao. For over 20 years, ao has been presumed to be a transcriptional regulator of core histones. However, the two existing ao mutant strains carry background mutations, and one of the strains is no longer available. We, therefore, created a CRISPR/Cas9-mediated knockout of the ao allele to separate the ao phenotype from background effects. Using the ∆ao flies, we found that although ao mutants exhibit maternal-effect lethality that is ameliorated by a reduction in histone copy number, ao does not affect histone transcript levels as previously reported. Given that Ao’s plant and mammalian orthologs are E3 ligases, we hypothesized that Ao is also an E3 ligase and used proteomics approaches to identify its potential targets. Finally, by maintaining the ∆ao flies as homozygous stocks, we allowed the strains to evolve de novo suppressors of the maternal-effect phenotype and used whole-genome sequencing to identify genomic changes associated with the loss of the ao maternal-effect lethality phenotype. Although our findings challenge the published molecular model of ao as a histone-transcript suppressor, we did find evidence for ao’s genetic interaction with histones. Understanding the basis of ao-associated maternal-effect lethality and its connection to histone copy number and heterochromatin remains an open and exciting question.