Characterizing modes of maternal RNA degradation during vertebrate development

描述（由申请人提供）：所有动物的早期发育最初依赖于母体提供的rna和蛋白质，这些rna和蛋白质由母体沉积到卵子中。受精卵的基因组最初转录不活跃，包括细胞分裂在内的所有细胞过程都是由这些初始的母体因素介导的。在后期阶段，合子基因组被激活并控制从母体贡献因子到新合成的合子rna和蛋白质的转换。随后发展的一个关键步骤是破坏母体成分。虽然这个过程已经知道了30多年，但在脊椎动物发育过程中清除母体指令所需的分子元素在很大程度上仍然未知。在本提案中，我将研究脊椎动物母体RNA降解的机制，以斑马鱼为模型系统。在目标1中，我将使用高通量测序来鉴定具有相关降解模式的母系衍生转录本。我将比较野生型胚胎和新生转录和小调控RNA合成被抑制的胚胎的时间过程表达数据，以确定在特定机制和特定时间控制下进行降解的转录本。在Aim 2中，我将分析母体转录本的3‘非翻译区（UTRs）的长度和序列内容，并在假设差异调节和降解是由这些序列介导的情况下，寻找合子转录本3’非翻译区之间的调节差异。在目标3中，我将使用计算方法和报告基因结构的组合来描述3' utr中存在的离散调控序列信号，这些信号是降解的决定因素。我希望共降解的母体rna也应该包含相同的信号集。综上所述，这些目标将阐明RNA转录物被差异降解的方式。尽管这些过程在发育过程中尤为突出，但它们与所有活细胞相关，这些活细胞需要维持一定数量的RNA信息以实现特定的表型。因此，了解RNA降解的机制是理解这一过程在发育障碍、癌症和疾病中是如何被错误调控的关键。

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.