The role of Spenito and m6A in establishing sexually dimorphic metabolism

项目总结 动物的性别决定途径使用基因表达电路将染色体差异放大到 男性和女性在解剖、神经和行为方面的差异。我们研究性别差异在 代谢表现为脂肪组织中脂肪储存的性别二型性。我们发现了二态表达 在果蝇主要脂肪组织中的性别决定基因，称为脂肪体(FB)， 这相当于哺乳动物的肝脏和脂肪组织。在这里，我们研究潜在的分子 果蝇幼虫储存脂肪的机制和影响，雄性比雌性储存更多的脂肪。我们的 实验室此前发现，Spenito(Nito)是一种保守的RNA结合蛋白，也是N6- 甲基腺苷(M6A)甲基转移酶复合体，无论性别，都是储存脂肪所必需的。FB- Nito的特定消耗消除了男性和女性之间的体脂差异，使两者相似 身体倾斜。我们发现，基于主调控基因剪接的典型性别决定途径 性致死(SXL)，在FB组织本身是活跃的，Nito是这种性别二型性所必需的 分子水平。已知Nito通过用m6A修饰SXL RNA在SXL剪接中发挥作用，但Nito在 培养细胞影响的基因是甲基转移酶其他成员缺失的两倍 很复杂。此外，我们发现，虽然FB特定击倒其他三个成员中的任何一个 复合体也使幼虫消瘦，性别差异基本保留。因此，我们建议 Nito m6A以性别特异性的方式甲基化FB中的脂肪调节RNA，以调节它们的剪接 和/或富足。我们将在两个具体目标上检验这一假设。在目标1中，我们将确定 男性和女性胎牛的代谢学和转录组差异是一种精确定位关键的方法 编码代谢酶的转录本，负责代谢性二型性。重复这一点 对患有Fb特异性Nito缺失的动物的分析将把列表缩小到Nito调控的mRNAs。初步 研究确定了多个令人信服的候选人。以确定性二型性是否自主起作用 在单个FB细胞内，或在FB作为一个组织内，我们将操纵SXL在雄性幼虫中的表达 在整个FB或在FB细胞的克隆内，分别使整个组织或细胞组“女性化”。我们 然后评估“女性化”细胞/组织中的脂肪储存情况。当Nito耗尽时重复这一分析将 测试对Nito的依赖。在目标2中，我们将在男性和女性中识别m6A修饰的FB mRNAs，并查看 当Nito耗尽时，这些m6A修饰如何变化，与其他变化相比 甲基转移酶亚基被耗尽。性二态、Nito依赖的M6A修饰的mRNAs将是 选择进行一项“拯救”实验，在雄性幼虫的FB中表达“雌性”版本，并询问这是否能够 “女性化”Fb新陈代谢。这项工作将揭示新陈代谢中性别差异的分子机制。

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.