The role of Spenito and m6A in establishing sexually dimorphic metabolism

Spenito 和 m6A 在建立性二态性代谢中的作用

• 批准号: 10604585
• 负责人: Arely Viridiana Diaz
• 金额: $ 3.61万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10604585
• 关键词: Adipose tissue; Affect; Anatomy; Animals; Behavioral; Binding; Body fat; Catabolism; Cells; Chromosomal Duplication; Clone Cells; Complex; Cultured Cells; Dependence; Drosophila genus; Drosophila melanogaster; Enzymes; Experimental Models; Family; Fat Body; Fatty acid glycerol esters; Female; Feminization; Gene Expression; Genes; Genetic; Goals; Gonadal Steroid Hormones; Gonadal structure; Hormones; Human; Individual; Larva; Lipids; Liver; Mammals; Messenger RNA; Metabolic; Metabolic Diseases; Metabolic Pathway; Metabolism; Methylation; Methyltransferase; Modification; Molecular; Monitor; Neurologic; Obesity; Organ; Ovary; Pathway interactions; Phenocopy; Predisposition; Protein Isoforms; RNA; RNA Splicing; RNA-Binding Proteins; Regulation; Regulator Genes; Research; Role; Sex Chromosomes; Sex Differences; Signal Transduction; System; Testing; Thinness; Tissues; Transcript; Translations; Wing; Work; dimorphism; experimental study; fly; gene function; glucose uptake; insight; knock-down; lipid metabolism; male; member; metabolic abnormality assessment; metabolic phenotype; metabolomics; overexpression; preservation; recruit; sex; sex determination; sexual dimorphism; tool; transcriptomics

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.

项目摘要 动物的性别决定途径使用基因表达回路来放大染色体差异， 男性和女性之间的解剖学、神经学和行为学差异。我们研究性别差异， 代谢表现为脂肪组织中脂肪储存的两性异形。我们发现双态表达 在果蝇的主要脂肪组织中，称为脂肪体（FB）， 相当于哺乳动物的肝脏和脂肪组织。在这里，我们研究了潜在的分子 机制和对果蝇幼虫脂肪储存的影响，其中雄性比雌性储存更多的脂肪。我们 实验室先前发现Spenito（Nito）是一种保守的RNA结合蛋白，也是N6- 甲基腺苷（m6 A）甲基转移酶复合物，无论性别，都需要适当的脂肪储存。FB- 特定的Nito消耗消除了男性和女性之间的体脂差异，使两者相似 瘦。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组织本身中是活跃的，Nito是在FB组织中这种性二型所必需的。 分子水平。已知Nito通过用m6 A修饰Sxl RNA而在Sxl剪接中起作用，但Nito缺失在Sxl剪接中起作用。 培养的细胞影响的基因是甲基转移酶其他成员耗竭的两倍 复杂.此外，我们发现，虽然FB特异性敲除其他三个成员中的任何一个， 复合体也使幼虫瘦，性别之间的差异大部分被保留下来。因此，我们建议 Nito m6 A以性别特异性方式甲基化FB中的脂肪调节RNA，以调节其剪接 和/或丰度。我们将在两个具体目标中检验这一假设。在目标1中，我们将确定 男性和女性FB之间的代谢组学和转录组学差异，作为一种精确定位关键的方法， 编码负责代谢性二型性的代谢酶的转录本。重复该 在具有FB特异性Nito耗竭的动物中的分析将把列表缩小到由Nito调节的mRNA。初步 研究确定了多个引人注目的候选人。为了确定两性异形是否是自主行为 在单个FB细胞内，或在作为组织的FB内，我们将操纵雄性幼虫中的Sxl表达， 在整个FB中或在FB细胞的克隆内，以分别使整个组织或细胞群“雌性化”。我们 然后将评估“雌性化”细胞/组织中的脂肪储存。当Nito耗尽时重复此分析将 测试Nito依赖性。在目标2中，我们将识别男性与女性中m6 A修饰的FB mRNA，并观察 当Nito耗尽时，这些m6 A修饰如何变化，与其他 甲基转移酶亚基被耗尽。性二态性、Nito依赖性m6 A修饰的mRNA将被 选择一个“救援”实验，以表达雄性幼虫FB中的“雌性”版本，并询问这是否能够 “女性化”FB代谢。这项工作将揭示新陈代谢性别差异的分子机制。