The role of Spenito and m6A in establishing sexually dimorphic metabolism

点击翻译按钮获取中文摘要

PROJECT SUMMARY Sex determination pathways in animals use gene expression circuits to amplify chromosomal differences into anatomic, neurological and behavioral distinctions between males and females. We study sex differences in metabolism manifested as sexual dimorphism in fat storage in adipose tissues. We found dimorphic expression of sex-determining genes in the major adipose tissue of Drosophila melanogaster, called the fat body (FB), which is equivalent to the liver and adipose tissue in mammals. Here we investigate the underlying molecular mechanisms and the effects on fat storage in Drosophila larvae, where males store more fat than females. Our lab previously found that Spenito (Nito), a conserved RNA-binding protein and a subunit of the N6- methyladenosine (m6A) methyltransferase complex, is required for proper fat storage regardless of sex. FB- specific depletion of Nito abolished body fat differences between males and females, making both similarly lean. We find that the canonical sex determination pathway, based on splicing of the master regulatory gene Sex lethal (Sxl), is active in the FB tissue itself, and that Nito is required for this sexual dimorphism at the molecular level. Nito is known to function in Sxl splicing by modifying Sxl RNA with m6A, but Nito depletion in cultured cells affects twice as many gene as does depletion of other members of the methyltransferase complex. Furthermore, we found that while FB-specific knockdown of any of three other members of the complex also made larvae lean, the differences between the sexes were mostly preserved. We thus propose that Nito m6A methylates fat-regulatory RNAs in the FB in a sex-specific manner to modulate their splicing and/or abundance. We will test this hypothesis in two specific aims. In Aim 1, we will determine the metabolomic and transcriptomic differences between male and female FBs, as a way of pinpointing key transcripts encoding metabolic enzymes responsible for the metabolic sexual dimorphism. Repeating this analysis in animals with FB-specific Nito depletion will narrow the list to mRNAs regulated by Nito. Preliminary studies identified multiple compelling candidates. To determine if the sexual dimorphism acts autonomously within individual FB cells, or within the FB as a tissue, we will manipulate Sxl expression in male larvae either throughout the FB or within clones of FB cells to “feminize” the entire tissue or groups of cells, respectively. We will then assess fat storage in the “feminized” cells/tissue. Repeating this analysis when Nito is depleted will test Nito dependence. In Aim 2, we will identify m6A-modified FB mRNAs in males versus females and see how these m6A modifications change when Nito is depleted, compared to changes when other methyltransferase subunits are depleted. Sexually dimorphic, Nito-dependent m6A-modified mRNAs will be chosen for a “rescue” experiment to express the “female” version in the FB of male larvae and ask if this is able to “feminize” FB metabolism. This work will uncover molecular mechanisms of sex differences in metabolism.