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

第一个哺乳动物 N 项的作用。

• 批准号: 8714415
• 负责人: Christine E Schaner-Tooley
• 金额: $ 23.41万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-05 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8714415
• 关键词: A Mouse; Acetylation; Advisory Committees; Affect; Aneuploidy; Antibodies; Apoptotic; Binding; Biological; Biological Assay; Biological Process; Biology; Cell Cycle; Cell Line; Cell Proliferation; Cell Separation; Cell physiology; Chromatin; Committee Members; Consensus Sequence; Cytoplasm; DNA; DNA Binding; DNA repair protein; DNA-Binding Proteins; Data; Defect; Development; Educational workshop; Elements; Embryo; Environment; Epithelial Cells; Exhibits; Fatty acid glycerol esters; Fibroblasts; Flow Cytometry; Fluorescence Resonance Energy Transfer; Fourier Transform; Future; Gene Combinations; Gene Targeting; Genes; Genetic; Gland; Glioblastoma; Goals; Growth; Harvest; Histology; Image; Immunoprecipitation; In Vitro; Knockout Mice; Learning; Mammary gland; Mass Spectrum Analysis; Measures; Mentors; Methods; Methylation; Methyltransferase; Microarray Analysis; Microscopy; Mitotic; Modeling; Modification; Monitor; Mono-S; Mus; Mutation; N-terminal; Neoplasm Metastasis; Nuclear; Oncogene Proteins; Operative Surgical Procedures; Paraffin Embedding; Patients; Pattern; Pharmaceutical Preparations; Phase; Phenotype; Post-Translational Protein Processing; Process; Proteins; Reagent; Research; Retinoblastoma Protein; Role; Running; Series; Site; Slide; System; Techniques; Testing; Therapeutic Agents; Tissue Sample; Training; Transgenic Organisms; Transplantation; Tumor Suppressor Genes; Tumor Suppressor Proteins; Tumor Tissue; Universities; Virginia; career development; cellular imaging; chromatin immunoprecipitation; design; in vitro Assay; in vivo; interest; leukemia; malignant breast neoplasm; mass spectrometer; mouse model; mutant; myosin light chain 2; protein function; research study; skills; stoichiometry; tool; tumor; tumor progression; tumorigenesis

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.

肿瘤发生涉及的基因列表非常广泛，但是它们的许多生物学功能 仍然未知。一个这样的基因，即mettl11a（现已更名为NRMT），已证明在 乳腺癌，但直到最近才被确定为第一个哺乳动物N末端甲基转移酶。 NRMT的降低还会导致多主轴表型，相关的非整倍性通常是 被认为有助于癌症进展。由于NRMT是一种新发现的蛋白质，因此目标的目标 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 它的正调如何导致肿瘤发生。前两个目标，确定N末端甲基化是否 是本构并确定N端甲基是否普遍改变其DNA的结合 底物旨在更好地了解N末端甲基化的基本生物学。这些目标会 涉及质谱，荧光激活细胞分选，FRET分析和敲除的构建 老鼠。第三个目的的目的是确定NRMT在发育和肿瘤发生中的作用。一个 如果多主轴NRMT敲低，将建立小鼠乳腺移植模型 表型导致发育缺陷和/或肿瘤发生。职业发展的关键要素 该建议的方面将是成功完成第三个目标所需的鼠标系统。 目标的实验是针对手术和腺体之间的停机时间设计的 肿瘤收获。 环境 弗吉尼亚大学有足够的能力完成本提案的所有三个目标。校园 咨询委员会成员唐·亨特（Don Hunt）博士的质谱设施有一个傅立叶变革质量 将N末端甲基化与乙酰化区分开所需的光谱仪。 UVA流式细胞仪 核心将为所有荧光激活细胞分选实验提供必要的训练。 UVA基因 靶向和转基因设施将创建NRMT敲除鼠标。研究组织学核心将 石蜡嵌入并制成所有正常小鼠组织和肿瘤的载玻片。我的咨询委员会成员博士 艾米·布顿（Amy Bouton）将有助于解释组织学。咨询委员会的专业知识和试剂 成员Todd Stukenberg博士将有助于表征NRMT多主轴表型。 UVA W.M.凯克蜂窝成像中心提供免疫荧光的最先进的成像设施 小鼠组织样品的成像和FRET分析中的训练。 Macara Lab目前有三名专家 小鼠生物学家可用于培训小鼠处理和手术技术。此外，UVA提供 许多旨在职业发展/培训的课程，包括UVA转基因方法和 应用研讨会，流式细胞仪训练研讨会，FRET显微镜的研讨会和A 博士后专业发展办公室提供的每月职业发展研讨会系列。