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Structure to phenotype analysis of a conserved RNA binding protein required for reproduction

繁殖所需的保守 RNA 结合蛋白的结构到表型分析

基本信息

批准号：
10693914
负责人：
Francesca Massi
金额：
$ 51.22万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10693914
关键词：
Affect Amino Acid Sequence Amino Acids Amyotrophic Lateral Sclerosis Animals Anterior Binding Biochemical Biochemistry Biological Biological Process CRISPR/Cas technology Caenorhabditis elegans Cell division Cells Child Computer Simulation Cytoplasm Data Defect Development Embryo Embryonic Development Event Family Family member Fertility Fertilization Gene Activation Gene Transfer Techniques Goals In Vitro Libraries Malignant Neoplasms Maps Maternal Messenger RNA Messenger RNA Methods Molecular Mothers Mutagenesis Mutate Mutation Myotonic Dystrophy NMR Spectroscopy Oocytes Oogenesis Orthologous Gene Pathway interactions Pattern Phenotype Post-Transcriptional Regulation Process Property Protein Dynamics Protein Family Proteins Publishing RNA RNA Binding RNA Sequences RNA Splicing RNA-Binding Proteins Regulation Relaxation Reporter Repression Reproduction Reproductive Biology Research Role Site-Directed Mutagenesis Specific qualifier value Specificity Structure Testing Transcript Translations Work Zinc Fingers biophysical techniques cell fate specification cell type developmental disease experimental study genome editing human disease in vivo innovation insight molecular dynamics oocyte maturation prevent protein structure segregation

项目摘要

During metazoan development, polarization of the body axes is of critical importance, as asymmetric cell division initiates cell specialization pathways. A family of conserved RNA-binding proteins characterized by CCCH-type tandem zinc finger (TZF) domains is required for axis polarization and cell type specification in the early embryo of C. elegans. A cascade of events essential to the oocyte- to-embryo transition is initiated by two members of this family, OMA-1/2. The process initiates with OMA-1/2 turnover and culminates with asymmetric segregation of cellular components to opposing poles of the embryo. Mutation of OMA-1/2 blocks oocyte maturation and prevents fertilization. We have observed that OMA-1 binds RNA with relatively low specificity in vitro, but regulates specific mRNAs in vivo. We have also determined that OMA-1 RNA binding is highly cooperative, a feature never before observed in a protein from the CCCH-type TZF family. Our goal is to investigate OMA- 1/2 activity from structure to phenotype. We will determine the molecular mechanism or RNA-binding cooperativity using NMR spectroscopy, computer simulations, and quantitative biochemistry. We will also define how RNA-binding cooperativity defines OMA-1 biological activity in worms. We will determine which mRNAs are regulated by OMA-1/2 in oocytes and of these mRNAs which are the most critical to reproduction. This research will lead to a new understanding of how the molecular properties of OMA-1 determine its function in reproductive biology in living animals.

在后生动物的发育过程中，体轴的极化是至关重要的，因为体轴不对称，细胞分裂启动细胞特化途径。一个保守的RNA结合蛋白家族其特征在于CCCH型串联锌指（TZF）结构域是轴极化所需的，细胞类型特化在C.优雅的一系列对卵母细胞至关重要的事件- 向胚胎的转变由该家族的两个成员OMA-1/2启动。该过程开始于 OMA-1/2周转，并以细胞成分不对称分离达到高潮，胚胎的两极。OMA-1/2突变阻断卵母细胞成熟并阻止受精。我们已经观察到OMA-1在体外以相对低的特异性结合RNA，但调节特异性RNA，体内mRNA。我们还确定OMA-1 RNA结合是高度合作的，以前从未在CCCH型TZF家族的蛋白质中观察到。我们的目标是调查OMA- 1/2活性从结构到表型。我们将确定分子机制或RNA结合利用核磁共振光谱学、计算机模拟和定量生物化学的协同性。我们将还定义了RNA结合协同性如何定义OMA-1在蠕虫中的生物活性。我们将确定卵母细胞中哪些mRNAs受OMA-1/2的调控，以及这些mRNAs中哪些是对繁殖至关重要这项研究将导致对分子如何 OMA-1的特性决定了其在活体动物生殖生物学中的功能。