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

描述 NSun 蛋白在小鼠胚胎发生开始时的作用

• 批准号: MR/N000080/1
• 负责人: Anthony Perry
• 金额: $ 83.69万
• 依托单位: University of Bath
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FN000080%2F1
• 关键词: Delineating; roles; NSun; proteins; onset

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hypothesis in the applicant laboratory, that carcinogenesis and embryogenesis share mechanisms of initiation.New unpublished evidence shows that most members of the NSun family are up-regulated immediately after fertilisation in the mouse.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.

受精作用将两个注定死亡的细胞--配子、精子和卵子--转化为一个细胞，从而产生一个完整的个体。这种转化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，我们对这一点的无知是值得注意的，这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。在正在进行的MRC资助的工作中，我们意外地发现哺乳动物（猪和小鼠）精子在受精过程中将NSun 1引入卵母细胞。这一初步发现是了不起的。NSun 1是一个小蛋白质家族的原型，该家族包括RNA甲基转移酶，尽管NSun家族成员的普遍重要性最近才被认识到，但很明显，它们参与多种发育途径，调节细胞分裂和增殖。这些是配子到胚胎转变中的关键事件，并强烈暗示与NSun的联系，这将加强申请人实验室中的过度增殖假说，即癌发生和胚胎发生共享启动机制。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。目前的建议旨在调查这一综合胚胎学和生物化学的方法，以产生一个模型的NSun功能在重新编程全能性在哺乳动物配子到胚胎transition.Our输出将包括仔细表征NSun转录本和蛋白质在小鼠受精过程中，并立即后，将显示如何干扰NSun表达在此期间影响胚胎发育。NSun具有高度保守的RNA甲基转移酶催化结构域，使我们能够采用我们的合作者最近开发的技术来捕获和固定NSun RNA底物。这将以单碱基分辨率揭示不同NSun的RNA底物，并使我们能够开始分析NSun靶标所起的早期胚胎作用。我们的工作通过其DNA结合能力鉴定了NSun 1，我们已经在活卵母细胞中证实了这一点，但NSuns的DNA结合迄今尚未报道。因此，我们将使用一种新的体内微珠测定法来检测卵母细胞和胚胎中NSun与DNA和RNA的结合;该测定法还将鉴定NSun中负责结合的结构域。总之，这项工作预计将表明母体mRNA如何在受精后立即进行调节，以及在其他情况下细胞效力变化是否共享调节机制。这些实验将揭示NSun中断的表型后果和哺乳动物发育开始时的靶RNA网络。更好地理解全能性出现中的NSuns有可能影响多能性的受控诱导，并有两个明确的额外翻译应用，我们将开始研究。首先，由于其促进增殖的作用，NSuns可能是致癌的。我们将与临床医生合作，调查是否有一个链接，可用于诊断和治疗癌症，特别是乳腺癌。其次，NSun活动的改变可能会损害生育能力。我们与一家大型IVF诊所的合作将确定NSuns是否代表（a）新的标志物，以及（B）生育能力受损的治疗靶点。

项目成果

Human embryonic genome activation initiates at the one-cell stage.

• DOI: 10.1016/j.stem.2021.11.012
• 发表时间: 2022-02-03
• 期刊: Cell stem cell
• 影响因子: 23.9
• 作者: Asami M;Lam BYH;Ma MK;Rainbow K;Braun S;VerMilyea MD;Yeo GSH;Perry ACF
• 通讯作者: Perry ACF

Genomic imprinting in mouse blastocysts is predominantly associated with H3K27me3.

• DOI: 10.1038/s41467-021-23510-4
• 发表时间: 2021-06-21
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Santini L;Halbritter F;Titz-Teixeira F;Suzuki T;Asami M;Ma X;Ramesmayer J;Lackner A;Warr N;Pauler F;Hippenmeyer S;Laue E;Farlik M;Bock C;Beyer A;Perry ACF;Leeb M
• 通讯作者: Leeb M

Intracytoplasmic Sperm Injection

胞浆内单精子注射

• DOI: 10.1007/978-3-319-70497-5_13
• 发表时间: 2018
• 作者: Griffin D

Switchable genome editing via genetic code expansion

通过遗传密码扩展进行可切换的基因组编辑

• DOI: 10.1101/349951
• 发表时间: 2018
• 作者: Suzuki T

Novel imprinted genes exemplify predominantly H3K27me3-dependent imprinting in mouse blastocysts

新型印记基因主要体现了小鼠囊胚中依赖于 H3K27me3 的印记

• 发表时间: 2021
• 期刊: Nature Communications
• 影响因子: 16.6
• 作者: Santini L.