Basonuclin and Ribosome Biogenesis in Mouse Oocyte and Embryo

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。母体mRNA在胚胎早期的翻译依赖于母体核糖体。然而，在卵母细胞成熟过程中，母体核糖体的很大一部分与许多其他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，而且似乎是浪费的。从核糖体生物学的主流观点来看，只有一种核糖体，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为这一困境提供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，其中一些用于卵子形成，而另一些用于翻译早期胚胎的母体mRNA。我们最近发现，核糖体RNA基因（rDNA）的细胞型特异性转录因子basonuclin （BNC1, BNC1）也是哺乳动物的母性效应基因。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7种变体rDNA （v-rDNA）亚型。随后对这些v- rnas的表征表明，每个变体都是独立的和组织特异性调节的。综上所述，这些结果表明，卵母细胞中rRNA亚群表达的扰动可能导致胚胎发育失败。我们的假设挑战了rDNA转录单位在所有组织中都是均匀和统一调节的主流观念。因此，我们的假设，如果正确的话，将修正目前对核糖体生物发生的理解，特别是它在卵子发生和早期胚胎发生中的含义。为了验证这一假设，我们提出了两个具体目标。特异性目标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。通过多序列比对，本研究将确定可变区域（标记）是否存在于rRNA之间，这表明rRNA具有异质性。特异性目的2将研究小鼠卵母细胞是否含有几种不同类型的rRNA，以及在卵母细胞成熟过程中是否有一些rRNA被选择性降解。我们将使用先前和Aim 1中鉴定的标记物来检查每个v-rDNA/rRNA亚型在oo细胞和早期胚胎中的表达。利用BNC1缺失的卵母细胞，我们将研究BNC1缺失是否会扰乱母体细胞中v-rDNA表达的平衡。这些研究将为在随后的R01应用中检验的假设提供实验基础。公共卫生相关性：最近的研究表明，“低质量”的卵子在很大程度上是导致女性不孕的原因，然而，什么是“好卵子”——母体储存的卵子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”的基本理解。具体来说，我们将研究母体rRNA储存是否具有异质性，以及这种异质性如何影响早期胚胎发育。这些信息可能最终转化为改进辅助生殖技术（ART）的实践。

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).