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 动物的性别确定途径使用基因表达回路将染色体差异扩增到 男性和女性之间的解剖学，神经和行为区别。我们研究性别差异 代谢在脂肪组织中的脂肪储存中表现为性二态性。我们发现二态表达 在果蝇的主要脂肪组织中，性别确定的基因称为脂肪体（FB）， 这等同于哺乳动物中的肝脏和脂肪组织。在这里，我们研究了基础分子 机制和对果蝇幼虫脂肪储存的影响，男性比女性储存更多的脂肪。我们的 实验室先前发现Spenito（Nito），保守的RNA结合蛋白和N6-的亚基 无论性别如何，都需要甲基腺苷（M6A）甲基转移酶复合酶复合物。 fb- NITO的特定定义消除了男性和女性之间的体内脂肪差异，这两者都类似 倾斜。我们发现，基于主调节基因的剪接的规范性确定途径 性致命（SXL）在FB组织本身中活跃，并且这种性二态性需要NITO 分子水平。已知NITO通过用M6A修改SXL RNA在SXL剪接中起作用，但NITO部署在 培养的细胞影响基因的两倍，是甲基转移酶其他成员的深度的两倍 复杂的。此外，我们发现，尽管FB特定的击倒了其他三名成员 复合物也使幼虫倾斜，性别之间的差异大部分被保留。因此，我们建议 NITO M6A以性别特异性的方式在FB中甲基化脂肪调节性RNA来调节其剪接 和/或抽象。我们将以两个具体的目的来检验这一假设。在AIM 1中，我们将确定 男性和雌性FBS之间的代谢组和转录组差异，以此来确定钥匙 编码代谢性二态性的代谢酶的转录本。重复这个 在具有FB特异性氮耗尽的动物中的分析将将清单范围缩小到由NITO调节的mRNA。初步的 研究确定了多个引人注目的候选人。确定性二态性是否自主起作用 在单个FB细胞中或在FB内作为组织，我们将操纵雄性幼虫的SXL表达 通过FB或FB细胞的克隆内部分别“使”整个组织或细胞组“女性化”。我们 然后，将评估“女性化”细胞/组织中的脂肪储存。重复此分析时，NITO耗尽将会 测试NITO依赖性。在AIM 2中，我们将确定男性与女性的M6A修饰的FB mRNA，并看到 与其他变化相比 甲基转移酶亚基耗尽。性二态，依赖NITO的M6A修饰的mRNA将是 选择“救援”实验以表达男性幼虫FB中的“女性”版本，并询问是否可以 “使” FB代谢“女性化”。这项工作将发现代谢中性别差异的分子机制。