Characterizing modes of maternal RNA degradation during vertebrate development

描述(申请人提供)：所有动物的早期发育最初依赖于母体的RNA和蛋白质的贡献，由母体沉积到卵子中。受精卵的基因组最初并不活跃地转录，所有的细胞过程，包括细胞分裂，都是由这些最初的母体因子调节的。在后来的阶段，合子基因组被激活，控制从母体贡献的因子切换到新合成的合子RNA和蛋白质。随后发展的一个关键步骤是销毁产妇组成部分。虽然这一过程已经知道了30多年，但在脊椎动物的发育过程中，清除母体指令所需的分子元件仍然基本上是未知的。在这项提议中，我将以斑马鱼为模型系统，研究脊椎动物母体RNA降解的机制。在目标1中，我将使用高通量测序来识别具有相关降解模式的母源转录本。我将比较野生型胚胎以及从头转录和小调节性RNA合成受到抑制的胚胎的时序表达数据，以确定在特定机制和特定时间控制下发生降解的转录本。在目标2中，我将分析母体转录本的3‘非翻译区(UTRs)的长度和序列含量，并在假设差异调控和降解是由这些序列介导的假设下，寻找合子转录本的3’UTRs之间的调控差异。在目标3中，我将使用计算方法和报告基因构建的组合来表征存在于3‘UTRs中的离散调控序列信号，这些信号是降解的决定因素。我预计，被共同降解的母体RNA也应该包含相同的信号集。这些目标将共同阐明RNA转录本的不同降解方式。虽然这些过程在发育过程中特别突出，但它们与所有活细胞有关，这些细胞需要维持受调控的RNA信息量，以实现特定的表型。因此，了解RNA降解的机制是理解这一过程如何在发育障碍、癌症和疾病中受到错误调控的关键。 与公共卫生相关：动物的早期发育依赖于对基因信息的严格时间控制，特别是哪些信息被允许用于产生蛋白质，哪些信息不再必要并被标记为销毁。虽然这种现象是胚胎发生的一个标志，在此期间胚胎对蛋白质的需求迅速变化，但实际上它对所有活细胞都是普遍存在的。因此，了解信息被破坏的机制以及标记它们被破坏的信号，对于理解和开发因基因信息量调节不当而导致的疾病(如癌症)的治疗方法是至关重要的。

DESCRIPTION (provided by applicant): Early development in all animals depends initially on the maternal contribution of RNAs and proteins, deposited by the mother into the egg. The genome of the zygote is not actively transcribed initially, and all cellular processes including cel division are mediated by these initial maternal factors. At a later stage, the zygotic genome is activated and control switches from maternally contributed factors to newly synthesized zygotic RNAs and proteins. A key step for subsequent development is the destruction of maternal components. While this process has been known for over three decades, the molecular elements required to clear maternal instructions during vertebrate development remain largely unknown. In this proposal, I will investigate the mechanisms of maternal RNA degradation in vertebrates, using zebrafish as a model system. In Aim 1, I will use high throughput sequencing to identify maternally derived transcripts with correlated degradation patterns. I will compare timecourse expression data in wildtype embryos as well as embryos in which de novo transcription and small regulatory RNA synthesis have been inhibited, in order to define transcripts undergoing degradation by specific mechanisms and under specific temporal control. In Aim 2, I will analyze the lengths and sequence content of the 3' untranslated regions (UTRs) of maternal transcripts and look for regulated differences between the 3'UTRs of zygotic transcripts, under the hypothesis that differential regulation and degradation is mediated by these sequences. In Aim 3, I will characterize the discrete regulatory sequence signals present in 3'UTRs that are the determinants of degradation, using a combination of computational methods and reporter gene constructs. I expect that maternal RNAs that are co-degraded should also contain the same sets of signals. Together these aims will elucidate the ways in which RNA transcripts are differentially degraded. Although these processes are especially prominent during development, they are relevant to all living cells, which need to maintain regulated quantities of RNA messages in order to achieve specific phenotypes. Thus, understanding the mechanisms underlying RNA degradation is key to understanding how this process is misregulated in developmental disorders, cancers, and disease. PUBLIC HEALTH RELEVANCE: Early development in animals depends on the strict temporal control of gene messages, specifically which messages are allowed to be used to generate proteins, and which are no longer necessary and marked for destruction. Although this phenomenon is a hallmark of embryogenesis, during which time the protein requirements of the embryo rapidly change, it is in fact ubiquitous to all living cells. Thus, understanding the mechanisms by which messages are destroyed, as well as the signals that mark them for destruction, is fundamental to understanding and developing treatments for diseases caused by the improper regulation of gene message quantities, such as cancer.