Basonuclin and Ribosome Biogenesis in Mouse Oocyte and Embryo

小鼠卵母细胞和胚胎中的基底核蛋白和核糖体生物发生

• 批准号: 7587729
• 负责人: RICHARD M SCHULTZ
• 金额: $ 19.69万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7587729
• 关键词: Affect; Animal Model; Area; Assisted Reproductive Technology; Biogenesis; Biology; Cloning; DNA Polymerase I; Data; Dependency; Development; Embryo; Embryonic Development; Equilibrium; Excision; Failure; Foundations; Future; Genes; Genetic Transcription; Genetic Translation; Genome; Heterogeneity; Human; Immunoglobulin Variable Region; Infertility; Investigation; Knockout Mice; Lead; Mammals; Maps; Maternal Messenger RNA; Mediating; Messenger RNA; Mus; Oocytes; Oogenesis; Pattern; Population; Population Heterogeneity; Reagent; Recombinant DNA; Regulation; Research Infrastructure; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Sequence Alignment; Signal Transduction; Staging; Structure; Techniques; Testing; Tissues; Translating; Translations; Variant; Woman; Zinc Fingers; base; basonuclin; cell type; egg; embryo cell; improved; interest; knock-down; null mutation; oocyte maturation; preimplantation; programs; public health relevance; rRNA Genes; transcription factor

DESCRIPTION (provided by applicant): An important component of the mammalian maternal store is ribosome. Translation of maternal mRNA in early embryo depends on the maternal ribosomes. However, a large portion of the maternal ribosomes is destroyed along with many other mRNAs during oocyte maturation. This programmed destruction of maternal ribosomes has been most perplexing and appears wasteful. From the prevailing view in ribosome biology, there is only one type of ribosome and why cannot early embryos use these ribosomes that are destroyed during oocyte maturation? Our recent results suggest an unconventional explanation to this dilemma. Based on these results, we hypothesize that oocytes transcribe several types of rRNA, some of which are used for oogenesis, whereas others for translating maternal mRNA in early embryos. We showed recently that basonuclin (BNC1, Bnc1), a cell-type specific transcription factor for the ribosomal RNA genes (rDNA), is also a mammalian maternal effect gene. Furthermore, our data suggest that BNC1 regulates a subset of rDNA. This observation led us to isolate seven subtypes of variant rDNA (v-rDNA). Subsequent characterization of these v-rDNAs showed that each variant is regulated independently and tissue-specifically. Taken together, these results suggest that perturbation of expression of a subset of rRNA in oocytes may lead to embryonic developmental failure. Our hypothesis challenges the prevailing notion that rDNA transcription units are homogeneous and regulated uniformly in all tissues. Therefore, our hypothesis, if correct, will revise current understanding of ribosomal biogenesis, especially its implication in oogenesis and early embryogenesis. To test this hypothesis, we propose two Specific Aims. Specific Aim 1 will determine if rRNA population is heterogeneous by cloning and examining the rRNA region (i.e., cytoplasmically expressed region) of each v-rDNA. Using multiple sequence alignment, this investigation will determine if variable regions (markers) exist among rRNAs, which indicate rRNA heterogeneity. Specific Aim 2 will investigate if mouse oocytes contain several different types of rRNA and whether some are selectively degraded during oocyte maturation. We will use markers identi- fied previously and in Aim 1 to examine the expression of each v-rDNA/rRNA subtypes in oo- cytes and early embryos. Using our BNC1-deficiency oocytes, we will investigate if loss of BNC1 perturbs the balance of v-rDNA expression in the maternal store. These studies will pro- vide experimental foundations for hypotheses to be tested in the ensuing R01 application. PUBLIC HEALTH RELEVANCE: Recent studies suggest that eggs of "low quality" are largely responsible for infertility in women, and yet what constitutes a "good egg"-of which the maternal store is an integral part-is unknown. The proposed studies will increase our basic understanding of what constitutes a "good egg". Specifically, we will examine if the maternal store of rRNA is heterogeneous and how this heterogeneity influences early embryonic development. Such information may ultimately translate to improving the practice of Assisted Reproductive Technology (ART).

描述（由申请人提供）：哺乳动物母体储存的一个重要组成部分是核糖体。早期胚胎中母体 mRNA 的翻译取决于母体核糖体。然而，在卵母细胞成熟过程中，大部分母体核糖体与许多其他 mRNA 一起被破坏。这种对母体核糖体的程序性破坏是最令人困惑的，而且似乎很浪费。从核糖体生物学的普遍观点来看，核糖体只有一种类型，为什么早期胚胎不能使用这些在卵母细胞成熟过程中被破坏的核糖体？我们最近的研究结果对这一困境提出了一种非常规的解释。基于这些结果，我们假设卵母细胞转录几种类型的 rRNA，其中一些用于卵子发生，而另一些用于在早期胚胎中翻译母体 mRNA。我们最近表明，基底核蛋白（BNC1，Bnc1）是核糖体 RNA 基因（rDNA）的细胞类型特异性转录因子，也是哺乳动物母体效应基因。此外，我们的数据表明 BNC1 调节 rDNA 的一个子集。这一观察结果使我们分离出了 rDNA 变异体 (v-rDNA) 的七种亚型。这些 v-rDNA 的后续表征表明，每个变体都是独立调节的且具有组织特异性。综上所述，这些结果表明，卵母细胞中 rRNA 子集表达的扰动可能会导致胚胎发育失败。我们的假设挑战了 rDNA 转录单位在所有组织中均质且受到统一调节的普遍观念。因此，我们的假设如果正确，将修正目前对核糖体生物发生的理解，特别是其在卵子发生和早期胚胎发生中的意义。为了检验这一假设，我们提出了两个具体目标。具体目标 1 将通过克隆和检查每个 v-rDNA 的 rRNA 区域（即细胞质表达区域）来确定 rRNA 群体是否异质。通过使用多重序列比对，本研究将确定 rRNA 之间是否存在可变区（标记），这表明 rRNA 异质性。具体目标 2 将研究小鼠卵母细胞是否含有几种不同类型的 rRNA，以及其中一些是否在卵母细胞成熟过程中选择性降解。我们将使用之前在目标 1 中鉴定的标记来检查卵母细胞和早期胚胎中每种 v-rDNA/rRNA 亚型的表达。使用我们的 BNC1 缺陷卵母细胞，我们将研究 BNC1 的缺失是否会扰乱母体储存中 v-rDNA 表达的平衡。这些研究将为随后的 R01 应用中待测试的假设提供实验基础。公共健康相关性：最近的研究表明，“低质量”的卵子是导致女性不孕的主要原因，但什么构成“好卵子”（母体储存是其中不可或缺的一部分）尚不清楚。拟议的研究将增加我们对什么是“好鸡蛋”的基本了解。具体来说，我们将检查母体储存的 rRNA 是否异质性以及这种异质性如何影响早期胚胎发育。此类信息最终可能会转化为改善辅助生殖技术（ART）的实践。