The role of the first mammalian N-term. methyltransferase, NRMT, in tumorigenesis

参与肿瘤发生的基因列表相当广泛，然而，它们的许多生物学功能 仍然未知。其中一个这样的基因，Mettl 11 a（现在改名为NRMT），已经被证明是低表达的， 乳腺癌，但最近才被确定为第一个哺乳动物N-末端甲基转移酶。 NRMT的减少也导致多纺锤体表型，并且相关的非整倍体通常是 被认为有助于癌症进展。由于NRMT是一种新发现的蛋白质， 建议是了解N-末端甲基化对蛋白质和细胞功能的作用，以便研究 它的失调是如何导致肿瘤发生的前两个目标是确定N端甲基化 是组成性的，并决定是否N-末端甲基化普遍改变其DNA结合， 底物，旨在更好地了解N-末端甲基化的基础生物学。这些目标将 包括质谱、荧光激活细胞分选、FRET分析和敲除构建 老鼠.第三个目标是确定NRMT在发育和肿瘤发生中的作用。一 将建立小鼠乳腺移植模型用于测定多纺锤体NRMT敲低是否 表型导致发育缺陷和/或肿瘤发生。职业发展的要素 这项建议的一个方面将是学习成功完成第三个目标所需的鼠标系统。 目标一和目标二的实验是针对手术和腺或 肿瘤收获。 环境 弗吉尼亚大学完全有能力完成这一提案的所有三个目标。校内 咨询委员会成员Don Hunt博士的质谱设备具有傅立叶变换质量 这是区分N末端甲基化和乙酰化所必需的光谱仪。UVA流式细胞术 核心将提供所有荧光激活细胞分选实验所需的培训。UVA基因 靶向和转基因设施将创建NRMT敲除小鼠。研究组织学核心将 石蜡包埋并制作所有正常小鼠组织和肿瘤的切片。我的顾问委员会成员博士。 Amy Bouton将协助解释组织学。咨询委员会的专业知识和试剂 成员托德Stukenberg博士将协助NRMT多纺锤体表型的表征。的UVA W.M.凯克细胞成像中心提供最先进的免疫荧光成像设备， 小鼠组织样品的成像和FRET分析中的训练。Macara实验室目前有三名专家 小鼠生物学家可提供小鼠处理和外科技术培训。此外，UVA提供 许多旨在职业发展/培训的课程，包括UVA转基因方法， 应用研讨会，流式细胞术培训研讨会，FRET显微镜研讨会，以及 由博士后专业发展办公室提供的每月职业发展研讨会系列。

The list of genes involved in tumorigenesis is quite extensive, however, many of their biological functions remain unknown. One such gene, Mettl11a (now renamed NRMT), has been shown to be under-expressed in breast cancers, but has only recently been identified as the first mammalian N-terminal methyltransferase. Reduction of NRMT also results in a multi-spindle phenotype, and the associated aneuploidy is often considered to contribute to cancer progression. As NRMT is a newly discovered protein, the goals of this proposal are to understand the role of N-terminal methylation on protein and cellular function, in order to study how its misregulation leads to tumorigenesis. The first two aims, determining whether N-terminal methylation is constitutive and determining whether N-terminal methylation universally alters the DNA binding of its substrates, are designed to better understand the basic biology of N-terminal methylation. These aims will involve mass spectrometry, fluorescent-activating cell sorting, FRET analysis, and construction of a knockout mouse. The objective of the third aim is to determine the role of NRMT in development and tumorigenesis. A mouse mammary transplant model will be established for assaying if the multi-spindle NRMT knockdown phenotype leads to developmental defects and/or tumorigenesis. The key element of the career development aspect of this proposal will be learning the mouse system needed for successful completion of the third aim. The experiments of aims one and two have been designed for the downtime between surgeries and gland or tumor harvesting. Environment The University of Virginia is well equipped for completion of all three aims of this proposal. The on-campus mass spectrometry facility, of advisory committee member Dr. Don Hunt, has a Fourier transform mass spectrometer necessary for distinguishing N-terminal methylation from acetylation. The UVA Flow Cytometry Core will provide the training necessary for all fluorescent-activating cell sorting experiments. The UVA Gene Targeting and Transgenic Facility will create the NRMT knockout mouse. The Research Histology Core will paraffin embed and make slides of all normal mouse tissue and tumors. My advisory committee member Dr. Amy Bouton will aid in interpretation of the histology. The expertise and reagents of advisory committee member Dr. Todd Stukenberg will aid in characterization of the NRMT multi-spindle phenotype. The UVA W.M. Keck Center for Cellular Imaging supplies state-of-the art imaging facilities for immunofluorescent imaging of mouse tissue samples and training in FRET analysis. The Macara lab currently has three expert mouse biologists available for training in mouse handling and surgical techniques. In addition, UVA offers numerous courses aimed at career development/training, including the UVA transgenic methods and applications workshop, the Flow Cytometry Training Workshop, the Workshop on FRET Microscopy, and a monthly career development seminar series offered by the Office of Postdoctoral Professional Development.