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

参与肿瘤发生的基因清单相当广泛，然而，它们的许多生物学功能 仍然不为人所知。其中一个这样的基因Mettl11a(现在被重新命名为NRMT)已经被证明在 乳腺癌，但直到最近才被确定为第一个哺乳动物的N末端甲基转移酶。 NRMT的减少也会导致多纺锤体表型，相关的非整倍体通常是 被认为是导致癌症进展的因素。由于NRMT是一种新发现的蛋白质，其目标是 建议了解N-末端甲基化对蛋白质和细胞功能的作用，以便研究 它的错误调控如何导致肿瘤的发生。前两个目标，确定N-末端甲基化 是构成的，并决定N-末端甲基化是否普遍改变其DNA结合 底物，旨在更好地理解N末端甲基化的基本生物学。这些目标将 包括质谱学、荧光激活细胞分类、FRET分析和构建基因敲除 老鼠。第三个目标是确定NRMT在发育和肿瘤发生中的作用。一个 将建立小鼠乳腺移植模型，用于检测多轴NRMT基因敲除情况 表型导致发育缺陷和/或肿瘤发生。职业发展的关键要素 这项提案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。 AIMS一和AIMS二的实验是为手术和腺体之间的停机时间设计的 肿瘤采集。 环境 弗吉尼亚大学做好了充分的准备，可以完成这项提案的所有三个目标。校园里的 咨询委员会成员唐·亨特博士的质谱学设备具有傅里叶变换质量 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 CORE将为所有荧光激活细胞分选实验提供必要的培训。UVA基因 靶向和转基因机构将创造NRMT基因敲除小鼠。组织学研究的核心意志 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Dr。 艾米·鲍顿将协助解释组织学。咨询委员会的专业知识和试剂 成员托德·斯图肯伯格博士将协助描述NRMT的多纺锤体表型。UVA W.M.凯克细胞成像中心为免疫荧光提供最先进的成像设备 小鼠组织样本的成像和FRET分析的培训。Macara实验室目前有三名专家 老鼠生物学家可以接受老鼠操作和外科技术方面的培训。此外，UVA还提供 许多旨在职业发展/培训的课程，包括UVA转基因方法和 应用工作坊、流式细胞术培训工作坊、FRET显微镜工作坊和 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.