ADAD1 and the post-meiotic male germ cell ribosome

ADAD1 和减数分裂后雄性生殖细胞核糖体

基本信息

批准号：
10707935
负责人：
Elizabeth M, Snyder
金额：
$ 7.85万
依托单位：
RUTGERS, THE STATE UNIV OF N.J.
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-21 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10707935
关键词：
Adolescent Alleles Antibodies Biogenesis Biology CRISPR/Cas technology Cell Differentiation process Cells Cellular biology Complex Coupled Cre driver Data Data Set Defect Development Enzymes Epitopes Foundations Gene Silencing Genetic Genetic Models Genome Germ Cells Goals Immunoprecipitation Knock-in Label Laboratories Lead Male Infertility Mass Spectrum Analysis Mediating Meiosis Messenger RNA Modeling Modification Molecular Molecular Mechanisms of Action Multiprotein Complexes Mutation Nuclear Outcome Phenotype Population Process Proteins Proteome RNA RNA Editing RNA-Binding Proteins Research RiboTag Ribosomal Proteins Ribosomal RNA Ribosomes Role Small Nucleolar RNA Spermatocytes Spermiogenesis Testing Testis Time Tissues Transcript Translational Regulation Translations Variant Work adenosine deaminase expectation genomic locus mRNA Translation male fertility mutant mutant mouse model novel null mutation protein expression tool transcriptome transcriptome sequencing translational impact

项目摘要

PROJECT SUMMARY / ABSTRACT Translation produces the proteins necessary for a cell’s function and relies on the ribosome, a highly conserved protein and RNA complex. Post-meiotic male germ cells are especially reliant on translation regulation, and consequently ribosome function. The long-term goal of this project is to determine the molecular mechanism by which ADAD1, a nuclear post-meiotic male germ cell-specific RNA binding protein, impacts translation. Previous work from our laboratory demonstrated Adad1 is required for male fertility as mutation leads to defects in post-meiotic germ cell differentiation. Although previously proposed as an RNA editing enzyme, our evidence demonstrates ADAD1 loss does not influence mRNA editing, making the molecular function of ADAD1 unknown. In preliminary data from our laboratory, we demonstrate ADAD1 loss leads to abnormal ribosome association of transcripts important for post-meiotic germ cell differentiation, reduced ribosomal RNA modification, and aberrant expression of ribosomal proteins and ribosome biogenesis factors. Together, these observations lead to the hypothesis that ADAD1 influences translation of post-meiotic transcripts by modulating nuclear ribosome biogenesis. The goal of this proposal is to generate the genetic tools and datasets necessary to robustly test this hypothesis through two aims. Aim 1 will define ADAD1 interacting proteins and RNAs in post-meiotic germ cells by generating an epitope tagged Adad1 allele and combining it with immunoprecipitation mass spectrometry and RNA sequencing. These efforts will shed light on ADAD1’s molecular mechanism of action. Aim 2 will quantify ribosome protein composition in Adad1 mutants and during germ cell development using quantitative mass spectrometry coupled to immunoprecipitation of a cell-specific epitope-tagged ribosome protein. Together, these analyses will quantify ADAD1’s impact on ribosome protein composition as well as define, for the first time, the meiotic and post-meiotic germ cell ribosome proteome. Historically, the ribosome was considered invariant across tissues and cells, however recent experimental evidence indicates ribosomal protein or RNA composition can vary, resulting in a specialized ribosome with unique translation dynamics. Whether germ cells leverage ribosome variation to control translation is an unexplored question that could have a dramatic impact on our understanding of germ cell biology. The studies proposed here will lay the foundations for defining the role of ADAD1 as well as identifying the male germ cell as a model for ribosome-level translation control.

项目概要/摘要翻译产生细胞功能所需的蛋白质，并依赖于核糖体，核糖体是一种高度保守的蛋白质和RNA复合物减数分裂后的雄性生殖细胞尤其依赖于翻译。该项目的长期目标是确定核糖体功能。 ADAD1（核减数分裂后雄性生殖细胞特异性 RNA 结合）的分子机制我们实验室之前的研究表明，Adad1 是男性所必需的。生育力作为突变导致减数分裂后生殖细胞分化的缺陷。提议作为 RNA 编辑酶，我们的证据表明 ADAD1 丢失不会影响 mRNA 编辑，使得 ADAD1 的分子功能未知。在我们实验室的初步数据中，我们证明 ADAD1 丢失会导致核糖体异常对减数分裂后生殖细胞分化很重要的转录物关联，核糖体 RNA 减少核糖体蛋白和核糖体生物合成因子的修饰和异常表达。这些观察结果得出这样的假设：ADAD1 影响减数分裂后的翻译该提案的目标是通过调节核核糖体生物发生来生成转录本。目标 1 将定义通过两个目标强有力地检验这一假设所需的遗传工具和数据集。 ADAD1 通过生成标记的表位与减数分裂后生殖细胞中的蛋白质和 RNA 相互作用 Adad1 等位基因并将其与免疫沉淀质谱和 RNA 测序相结合。将阐明 ADAD1 的分子作用机制 Aim 2 将量化核糖体。使用定量质量分析 Adad1 突变体和生殖细胞发育过程中的蛋白质组成光谱测定法与细胞特异性表位标记核糖体蛋白的免疫沉淀相结合。总之，这些分析将量化 ADAD1 对核糖体蛋白质组成以及首次定义减数分裂和减数分裂后生殖细胞核糖体蛋白质组。从历史上看，核糖体被认为在组织和细胞中是不变的，但最近实验证据表明核糖体蛋白或 RNA 组成可能会发生变化，从而导致具有独特翻译动力学的特化核糖体。生殖细胞是否利用核糖体变异。控制翻译是一个尚未探索的问题，可能会对我们的理解产生巨大影响这里提出的研究将为将 ADAD1 的作用定义为奠定基础。以及将雄性生殖细胞识别为核糖体水平翻译控制的模型。