Delineating the roles of NSun proteins at the onset of mouse embryogenesis

受精将两个注定要死亡的细胞--配子、精子和卵子--转变为一个完整的个体。这种转变以我们难以理解的方式整合了复杂的细胞内事件。考虑到配子到胚胎转变的基本性质及其对健康和疾病(包括干细胞和癌症)的影响，我们对此的忽视是值得注意的，并迫使我们开发一个全面的模型来解释这种转变是如何发生的。在MRC资助的正在进行的工作中，我们有了一个意想不到的发现，哺乳动物(猪和小鼠)的精子在受精过程中将NSun1引入卵母细胞。这一初步发现是值得注意的。NSun1是包括RNA甲基转移酶在内的一个小蛋白家族的原型，尽管NSun家族成员的重要性直到最近才得到重视，但很明显，它们参与了多种发育途径，调节细胞分裂和增殖。这些都是配子到胚胎转变的关键事件，并强烈暗示与NSun的联系，这将加强申请实验室中的总体假说，即癌症发生和胚胎发生具有共同的启动机制。新的未发表的证据表明，NSun家族的大多数成员在小鼠受精后立即上调。本提案试图通过胚胎学和生物化学的综合方法来研究这一点，以产生一个NSun在哺乳动物配子到胚胎转变过程中重新编程为全能性的模型。我们的成果将包括在小鼠受精期间和受精后立即仔细分析NSun转录本和蛋白质的特征，并将展示在这一时期干扰NSun表达如何影响胚胎发育。NSun具有高度保守的RNA甲基转移酶催化域，使我们能够利用我们的合作者最近开发的技术来捕获和表征NSun RNA底物。这将揭示不同NSun在单碱基分辨率下的RNA底物，并使我们能够开始分析NSun靶标所扮演的早期胚胎角色。我们的工作是通过NSun1的DNA结合能力来鉴定NSun1，我们已经在活的卵母细胞中证实了这一点，但NSuns与DNA的结合到目前为止还没有报道。因此，我们将使用一种新的体内微球实验来表征卵母细胞和胚胎中的NSun与DNA和RNA的结合；该实验还将识别NSun中负责结合的结构域。综上所述，这项工作有望表明母体mRNA在受精后立即受到调控，以及调控机制是否与其他环境中细胞潜能的变化相同。这些实验将揭示NSun中断的表型后果，并在哺乳动物发育开始时针对RNA网络。更好地理解NSun在全能的出现中，有可能通过两个明确的额外翻译应用来影响多能性的受控诱导，我们将开始研究这两个明显的翻译应用。首先，由于其促进增殖的作用，NSun可能是致癌的。我们将与临床医生合作，调查是否有一种联系可以用于癌症，特别是乳腺癌的诊断和治疗。其次，NSun活动的改变可能会损害生育能力。我们与一家主要的试管受精诊所的合作将确定NSun是否代表(A)新的标记物，(B)生育力受损的治疗靶点。

Fertilisation transforms two cells that are destined to die - the gametes, sperm and egg - into one that engenders an entire individual. The transformation integrates complex intracellular events in ways that we hardly understand. Our ignorance of this is remarkable given the fundamental nature of the gamete-to-embryo transition and its implications for health and disease - including stem cells and cancer - and compels us to develop a comprehensive model explaining how the transition occurs.In ongoing MRC-funded work, we made the unexpected discovery that mammalian (porcine and mouse) sperm introduce NSun1 into oocytes during fertilisation. This preliminary finding is remarkable. NSun1 is the prototype of a small protein family that includes RNA methyl-transferases and although the general importance of NSun family members is only recently becoming appreciated, it is clear that they are involved in multiple developmental pathways, regulating cell division and proliferation. These are key events in the gamete-to-embryo transition and strongly imply a link to NSuns that would strengthen the over-arching hypothesis in the applicant laboratory, that carcinogenesis and embryogenesis share mechanisms of initiation.New unpublished evidence shows that most members of the NSun family are up-regulated immediately after fertilisation in the mouse. The present proposal seeks to investigate this in an integrated embryological and biochemical approach to produce a model of NSun function in reprogramming to totipotency during the mammalian gamete-to-embryo transition.Our output will include careful characterisation of NSun transcripts and proteins during and immediately after mouse fertilisation and will show how interfering with NSun expression during this period affects embryo development. NSuns have a highly-conserved RNA methyl-transferase catalytic domain, enabling us to employ a technique recently developed by our collaborator to capture and characterise NSun RNA substrates. This will reveal the RNA substrates of different NSuns at single-base resolution and allow us to begin an analysis of early embryonic roles played by NSun targets. Our work identified NSun1 by its DNA binding ability, which we have confirmed in living oocytes, but DNA binding by NSuns has not so far been reported. We shall therefore characterise the binding of NSuns in oocytes and embryos to DNA and RNA using a novel in vivo microbead assay; the assay will also identify the domains in NSuns responsible for binding. Together, this work is expected to indicate how maternal mRNA are regulated immediately after fertilisation and whether regulatory mechanisms are shared by cellular potency changes in other contexts.These experiments will reveal the phenotypic consequences of NSun disruption and target RNA networks at the onset of mammalian development. A better understanding of NSuns in the emergence of totipotency has the potential to impact the controlled induction of pluripotency with two clear additional translational applications that we will start to investigate. First, because of their roles promoting proliferation, NSuns may be oncogenic. We will work with clinicians to investigate whether there is a link that can be used in the diagnosis and treatment of cancer, particularly breast cancer. Secondly, altered NSun activity may impair fertility. Our collaboration with a major IVF clinic will determine whether NSuns represent (a) novel markers, and (b) treatment targets of impaired fertility.