Coordinating Spindle Assembly and Meiotic Maturation via Regulation of RNA

通过 RNA 调节协调纺锤体组装和减数分裂成熟

基本信息

批准号：
8540171
负责人：
Matthew Simon Cook
金额：
$ 3.99万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-01-01 至 2015-06-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8540171
关键词：
Address Biological Assay Biology Cell Cycle Cell Cycle Progression Cell Nucleus Cell division Cell physiology Cells Chromosome Segregation Chromosomes Cytoplasm Data Development Embryo Embryonic Development Ensure Event Fertilization Fluorescence Resonance Energy Transfer Gene Expression Profile Genetic Genetic Models Genetic Transcription Genome Germ Germ Cells Germ cell tumor Haploidy Human Image In Vitro Knockout Mice Lead Life Link Luciferases Meiosis Messenger RNA Mitotic spindle Mus Oocytes Pathway interactions Pluripotent Stem Cells Post-Transcriptional Regulation Pregnancy Process RNA RNA-Binding Proteins Regulation Reproductive Health Role Small RNA Somatic Cell Staging Sterility Structure System Tertiary Protein Structure Testing Time Transcript Translations Work Xenopus base cell type developmental disease egg human DICER1 protein insight knock-down mutant novel nuclear division oocyte maturation overexpression research study sperm cell stem cell population zygote

项目摘要

DESCRIPTION (provided by applicant): In humans, the phenomenon of cellular reprogramming occurs naturally in a single cell type: the germ cell. At fertilization, the haploid nuclei of a sperm and egg fuse and are reprogrammed by the cytoplasm of the mature oocyte to form a totipotent zygote. Amazingly, this process occurs in the absence of RNA transcription and is inextricably linked to successful spindle assembly and nuclear division during the final stages of meiosis. Thus, post-transcriptional regulation of the transcriptome is paramount to ensure successful spindle assembly prior to completing the oocyte-to-zygote transition. Recent studies in mouse have revealed a critical role at this stage for endogenous-siRNAs (endo-siRNAs) and the RNA binding protein DAZL (which localizes to the spindle poles) in promoting proper spindle assembly. However, it remains unclear how they cooperate to directly regulate this process. Preliminary data suggest that DAZL and endo-siRNAs both regulate transcripts related to the RanGTP pathway, a critical player in the formation of the mitotic spindle. Therefore, I hypothesize that DAZL and endo-siRNAs work together to spatially regulate translation of target mRNAs that are critical for meiotic spindle organization and cell cycle progression. To address this hypothesis, genetic fusion constructs with various deletions of protein domains within DAZL combined with live oocyte imaging during maturation will be used to determine the mechanism and importance of DAZL localization to the meiotic spindle. In addition, mRNA targets of DAZL will be immunoprecipitated and studied to determine the downstream effects of DAZL's role on formation of the meiotic spindle, particularly with regards to the levels of RanGTP. Finally, the connection between DAZL and endo-siRNAs will be explored through their connection to the RanGTP pathway. Various luciferase constructs, FRET probes, knockdown and overexpression assays, as well as small RNA-deficient (Dicer mutant) oocytes will be used to determine whether endo-siRNAs directly regulate Rangap1, and thus RanGTP levels, to facilitate normal spindle assembly. The experiments detailed in this proposal will elucidate how an RNA binding protein, DAZL, and endo-siRNAs work together to coordinately regulate spindle assembly and, thus, cell fate. This work will also provide novel insights for better understanding other pluripotent stem cell populations, including the induced reprogramming of somatic cells to pluripotent cells.

描述（由申请人提供）：在人类中，细胞重编程现象自然发生在单一细胞类型中：生殖细胞。受精时，精子和卵子的单倍体细胞核融合，并由成熟卵母细胞的细胞质重新编程，形成全能合子。令人惊讶的是，这个过程在没有 RNA 转录的情况下发生，并且与减数分裂最后阶段成功的纺锤体组装和核分裂有着千丝万缕的联系。因此，转录组的转录后调控对于确保在完成卵母细胞到受精卵的转变之前成功完成纺锤体组装至关重要。最近对小鼠的研究表明，内源 siRNA (endo-siRNA) 和 RNA 结合蛋白 DAZL（定位于纺锤体极）在此阶段在促进纺锤体正常组装方面发挥着关键作用。然而，目前尚不清楚他们如何合作来直接监管这一过程。初步数据表明，DAZL 和内切 siRNA 均调节与 RanGTP 途径相关的转录物，RanGTP 途径是有丝分裂纺锤体形成的关键参与者。因此，我假设 DAZL 和内切 siRNA 共同作用，在空间上调节目标 mRNA 的翻译，这对于减数分裂纺锤体组织和细胞周期进展至关重要。为了解决这一假设，DAZL 内蛋白质结构域的各种删除与成熟过程中的活卵母细胞成像相结合的基因融合构建体将用于确定 DAZL 定位到减数分裂纺锤体的机制和重要性。此外，DAZL 的 mRNA 靶点将进行免疫沉淀和研究，以确定 DAZL 对减数分裂纺锤体形成的下游影响，特别是 RanGTP 水平。最后，DAZL 和内切 siRNA 之间的联系将通过它们与 RanGTP 途径的连接来探索。各种荧光素酶构建体、FRET 探针、敲低和过表达测定以及小 RNA 缺陷（Dicer 突变体）卵母细胞将用于确定内切 siRNA 是否直接调节 Rangap1，从而调节 RanGTP 水平，以促进正常纺锤体组装。该提案中详述的实验将阐明 RNA 结合蛋白、DAZL 和内切 siRNA 如何协同调节纺锤体组装，从而协调细胞命运。这项工作还将为更好地理解其他多能干细胞群提供新的见解，包括诱导体细胞重编程为多能细胞。