Functional consequences of disrupting RNA-binding protein function for oogenesis and oocyte-to- embryo transition.

基因表达的严格控制对生命至关重要，并通过转录和转录后调节机制的组合来实现。然而，在配子发生和受精后发育的某些阶段，转录基本上是无活性的，这意味着基因表达完全由转录后机制控制。这涉及通过RNA结合蛋白激活、抑制或破坏细胞质中预先存在的mRNA。最近的研究表明，哺乳动物编码超过1000种功能性RNA结合蛋白，但尽管这一大类蛋白质与包括生殖、代谢、神经和致癌疾病在内的广泛疾病之间存在关键联系，但有关其功能的信息仅可用于少数RNA结合蛋白家族。这些包括DAZ家族，其中DAZL是研究得最好的，并且是各种模式生物中雄性和雌性配子发生所需的（PMID：19225045中综述），并且在哺乳动物的卵母细胞向胚胎转变中也具有额外的作用（PMID：21460039）。在这里，我们的目标是探索在生育能力降低的人类患者中鉴定的DAZL突变的功能后果。（卵巢功能不全，精子发生缺陷）对减数分裂和向有丝分裂过渡所需的mRNA的调节。我们的初步数据使我们相信这些突变可能是致病的，本项目的第一个目的是将这些数据扩展到小鼠卵母细胞和胚胎中，以确定它们是否足以破坏卵子发生和卵母细胞向胚胎的转变。第二个目的是了解DAZL中的不同突变如何破坏其在调节mRNA中的不同功能（PMID：16278232，16001084，17526644，未发表），重点是其与其他关键RNA结合蛋白家族的相互作用。本项目将涉及多种分子生物学技术和先进的辅助生殖技术（如小鼠卵母细胞的分离、体外成熟和体外受精，以及显微操作等）。

Tight control of gene expression is critical to life and is achieved by a combination of transcriptional and post-transcriptional regulatory mechanisms. However during certain stages of gametogenesis and post-fertilisation development, transcription is essentially inactive meaning that gene expression is controlled exclusively by post-transcriptional mechanisms. This involves the activation, repression or destruction of pre-existing mRNAs in the cytoplasm by RNA-binding proteins. Recent studies have revealed that mammals encode over 1000 functional RNA-binding proteins but despite critical links between this large class of proteins and a wide range of diseases including reproductive, metabolic, neurological and oncogenic disorders, information on their functions is only available for a handful of RNA-binding protein families. These include the DAZ family of which DAZL is the best studied and is required for male and female gametogenesis in a variety of model organisms (reviewed in PMID: 19225045) and also has an additional role in the oocyte to embryo transition in mammals (PMID: 21460039).Here we aim to explore the functional consequences of DAZL mutations identified in human patients with reduced fertility (ovarian insufficiency, defective spermatogenesis) on the regulation of mRNAs required for meiosis and the transition to mitosis. Our preliminary data leads us to believe that these mutations may be causative and the first aim of this project is to extend these data into mouse oocytes and embryos to determine whether they are sufficient to disrupt oogenesis and oocyte-to-embryo transition. The second aim is to understand how different mutations in DAZL disrupt its different functions in regulating mRNAs (PMID: 16278232, 16001084, 17526644, unpublished) focusing on its interactions with other key RNA-binding protein families. This project will involve a wide variety of molecular biology techniques and advanced Assisted Reproductive Technologies (e.g. isolation, in vitro maturation and in vitro fertilisation of mouse oocytes, and micromanipulation etc.).