RUI: Pokeweed Antiviral Protein selection of mRNA; Effects of mRNA structure and initiation factors

RUI：美洲商陆抗病毒蛋白mRNA的选择；

• 批准号: 0919626
• 负责人: lawrence kobilinsky
• 金额: $ 41.57万
• 依托单位: CUNY John Jay College of Criminal Justice
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0919626&HistoricalAwards=false
• 关键词: RUI; Pokeweed; Antiviral; Protein; selection

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Plant pathogens (viral, fungal, bacterial, or pest) affect a significant number of food crops worldwide, often indiscriminately. The impact is considerable and far-reaching, particularly as these agents have numerous invasion strategies. One important plant defense mechanism employs ribosome-inactivating proteins (RIPs), which are naturally occurring cytotoxic agents. Their biochemical activity is to depurinate the universally conserved sarcin/ricin loop (SRL) of rRNA, resulting in the cessation of protein synthesis in the infected cells. Pokeweed Antiviral Protein (PAP) is a type I RIP with broad-spectrum antiviral activity against plant and animal viruses. In addition to the canonical rRNA depurination activity, PAP also possesses mRNA recognition, cap binding, and depurination activities that are distinct from other RIPs. Thus, PAP has demonstrated dual access to the translation machinery and is a model for agents that disrupt eukaryotic translation. The goal of this project is to characterize the mechanism by which PAP selects RNA targets for depurination. The PI will employ a combination of biophysical and biochemical approaches to (1) determine the extent and affinity of eukaryotic initiation factor (eIF) participation in selection of RNA and (2) identify the essential mRNA structural elements that affect PAP affinity and enzymatic activity. Oligonucleotides of capped and uncapped mRNA with differing secondary structure will be used in this study to elucidate these structural requirements. This research will increase our understanding of viral infections and how they affect protein synthesis, potentially leading to new anti-viral approaches. Such insight into plant defense mechanisms will guide practical strategies to reduce crop losses due to pathogen infection. Broader Impacts: This research will be performed primarily by undergraduate science majors at John Jay College of the City University of New York (CUNY). CUNY is the nation's largest urban public university system, serving over 450,000 students. Notably, the training will be conducted on a campus that enrolls more than 60% Hispanic and African American students and is the largest Hispanic-Serving Institution in the northeastern U.S. and the fourth largest in the nation. During her first five years in the Department of Sciences, the PI has trained a total of 22 students in her research laboratory; this number includes 12 undergraduates. The majority of these students have been of a minority demographic and all but two have been women. The educational impact of this project includes training in modern biophysical and biochemical techniques that are an extension of laboratory classroom skills. The research includes approaches that are suitable for the involvement of students at many levels (high school, undergraduate, graduate, and post-graduate), thus providing comprehensive training while producing scientifically literate citizens. Upon completion of their training, students will be well-equipped for advanced degree programs and/or careers in the STEM fields. The research relies heavily on peers as co-researchers in its approach; thus cooperativity and teamwork skills are naturally incorporated into the students' training. In addition to providing research opportunities for undergraduates, this project allows participating students to travel annually to a national conference. This will give them an opportunity to interact with the broader scientific community, to present experimental results to other researchers, to network with peers from other institutions, and meet with prospective graduate school representatives. Through this project, the students will learn how high quality basic research contributes to larger areas of science and society such as improved agricultural practices, more robust agricultural crops, secure food supply protection, and the nation's economy.

该奖项是根据2009年美国复苏和再投资法案(公法111-5)资助的。植物病原体(病毒、真菌、细菌或害虫)通常不分青红皂白地影响世界各地的大量粮食作物。影响是相当大和深远的，特别是在这些特工有许多入侵策略的情况下。一种重要的植物防御机制是利用核糖体失活蛋白(RIPs)，这是一种自然产生的细胞毒剂。它们的生化活性是去除rRNA中普遍保守的sarcin/ricin环(SRL)，导致感染细胞蛋白质合成停止。商陆抗病毒蛋白(PAP)是一种具有广谱抗植物和动物病毒活性的I型RIP。PAP除了具有典型的rRNA去嘌呤活性外，还具有不同于其他RIPs的信使核糖核酸识别、帽结合和去嘌呤活性。因此，PAP已经证明了对翻译机制的双重访问，并且是扰乱真核翻译的代理人的模型。这个项目的目标是描述PAP选择RNA目标进行去嘌呤的机制。PI将采用生物物理和生化方法相结合的方法来(1)确定真核细胞起始因子(EIF)参与RNA选择的程度和亲和力，以及(2)确定影响PAP亲和力和酶活性的基本mRNA结构元件。在这项研究中，将使用具有不同二级结构的封顶和未封顶的mRNA的寡核苷酸来阐明这些结构要求。这项研究将增加我们对病毒感染及其如何影响蛋白质合成的理解，可能导致新的抗病毒方法。对植物防御机制的这种洞察将指导减少病原菌侵染造成的作物损失的实用策略。更广泛的影响：这项研究将主要由纽约城市大学(CUNY)约翰·杰伊学院(John Jay College)的本科生进行。CUNY是美国最大的城市公立大学系统，为超过45万名学生提供服务。值得注意的是，培训将在一个招收60%以上拉美裔和非裔美国人学生的校园内进行，该校是美国东北部最大的拉美裔服务机构，也是美国第四大机构。在她进入科学系的头五年里，PI在她的研究实验室总共培训了22名学生；这个数字包括12名本科生。这些学生中的大多数都是少数族裔，除两人外，其余都是女性。该项目的教育影响包括现代生物物理和生化技术方面的培训，这些技术是实验室课堂技能的延伸。这项研究包括适合多个层次(高中、本科生、研究生和研究生)的学生参与的方法，从而在提供全面培训的同时培养具有科学素养的公民。完成培训后，学生将为STEM领域的高级学位课程和/或职业生涯做好准备。这项研究在很大程度上依赖于同伴作为合作研究人员；因此，合作精神和团队合作技能自然被纳入到学生的培训中。除了为本科生提供研究机会外，该项目还允许参加该项目的学生每年前往参加全国会议。这将使他们有机会与更广泛的科学界互动，向其他研究人员展示实验结果，与其他机构的同行建立网络，并会见未来的研究生院代表。通过这个项目，学生们将了解高质量的基础研究如何为更广泛的科学和社会领域做出贡献，如改进农业实践、更茁壮的农作物、确保粮食供应保护和国家经济。