Selection of the 3' end mRNA processing site by Cleavage Factor I

通过切割因子 I 选择 3 末端 mRNA 加工位点

• 批准号: 1122768
• 负责人: Alex (Andrew) Bohm
• 金额: $ 80.56万
• 依托单位: Tufts University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2015-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1122768&HistoricalAwards=false
• 关键词: Selection; end; mRNA; processing; site

Intellectual merit: Gene expression is the process of synthesizing functional proteins using information stored in the nucleus as DNA and transmitted to cellular sites of protein synthesis in the form of mRNA molecules, produced by copying (transcribing) DNA. An essential step in gene expression is the precise trimming of the 3'-ends of messenger RNA (mRNA) molecules by 3'-end processing enzymes that recognize specific RNA sequences. This process determines the length of each mRNA molecule and affects the efficiency with which it is utilized as a template for protein synthesis. Roughly half of human genes contain more than one potential site of 3'-end processing and are subject to alternative processing. Selection of the appropriate processing site is particularly important in the synthesis of proteins involved in immune responses and in cell differentiation. This project uses yeast as a model system to study the mechanism of 3'-end processing and focuses on an evolutionarily conserved complex of five proteins called Cleavage Factor I (CF I) that recognizes mRNA signal sequences which define the sites of 3'-end processing. The research will utilize single-particle electron microscopy, coupled with RNA foot-printing and molecular modeling techniques to construct models of the overall structure of CF I and elucidate how it recognizes mRNA signal sequences and directs processing of the 3'-ends of mRNAs. Yeast genetics, coupled with biochemical binding experiments, will be used to verify and refine the resulting molecular models. This work is of fundamental importance because mRNA 3'-end processing affects the expression of all proteins in all eukaryotic cells. Broader impacts: The project will provide hands-on research training for the graduate students in the Tufts Biochemistry program, postdoctoral fellows and summer trainees, including undergraduates who will be recruited through an NIH-sponsored program entitled "Building Diversity in the Biomedical Sciences" and high school students who will come to the laboratory through the Teachers and High School Students Program administered by the Tufts Office of Minority Affairs. The funds will also permit Dr. Bohm to continue his mentorship activities within the Tufts Biochemistry Program and within the Tufts PREP program, a postbaccalaureate program that prepares students from underrepresented groups for graduate school. In addition, the techniques used in this work will be integrated into two courses Dr. Bohm currently teaches. The first is a graduate course on molecular recognition. The second is a summer workshop-style classroom/laboratory course designed to introduce participants in the use of scattering methods to analyze macromolecular complexes in their own research. Thus, this course is a particularly important mechanism for disseminating the technical aspects of the research and helping others apply these methods to other biological systems.

智力优势：基因表达是利用储存在细胞核中的DNA信息合成功能性蛋白质的过程，并通过复制（转录）DNA以mRNA分子的形式传递到蛋白质合成的细胞位点。基因表达的一个重要步骤是信使RNA （mRNA）分子的3'端加工酶识别特定RNA序列的精确修剪。这个过程决定了每个mRNA分子的长度，并影响其作为蛋白质合成模板的效率。大约一半的人类基因包含一个以上的3'端加工的潜在位点，并受到替代加工。选择合适的加工位点对于参与免疫反应和细胞分化的蛋白质合成尤为重要。本项目以酵母为模型系统研究3‘端加工机制，重点研究一种进化上保守的五种蛋白质复合物，称为切割因子I (cfi)，它识别定义3’端加工位点的mRNA信号序列。本研究将利用单粒子电子显微镜，结合RNA足迹和分子建模技术构建CF I的整体结构模型，阐明其如何识别mRNA信号序列并指导mRNA的3'端加工。酵母遗传学，结合生化结合实验，将用于验证和完善所得的分子模型。这项工作具有重要的基础意义，因为mRNA 3'端加工影响所有真核细胞中所有蛋白质的表达。更广泛的影响：该项目将为塔夫茨大学生物化学项目的研究生、博士后和暑期实习生提供实践研究培训，包括通过美国国立卫生研究院资助的“生物医学科学建设多样性”项目招募的本科生，以及通过塔夫茨大学少数民族事务办公室管理的教师和高中生项目来到实验室的高中生。这笔资金还将允许Bohm博士继续他在塔夫茨生物化学项目和塔夫茨PREP项目中的指导活动，这是一个为来自代表性不足群体的学生准备研究生院的学士学位后项目。此外，这项工作中使用的技术将整合到Bohm博士目前教授的两门课程中。第一个是关于分子识别的研究生课程。第二个是暑期工作坊式的课堂/实验课程，旨在向参与者介绍在他们自己的研究中使用散射方法来分析大分子复合物。因此，本课程是传播研究的技术方面和帮助他人将这些方法应用于其他生物系统的特别重要的机制。