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

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）影响世界范围内大量的粮食作物，通常不分青红皂白。影响是相当大的和深远的，特别是因为这些病原体有许多入侵策略。一种重要的植物防御机制是利用核糖体失活蛋白（RIPs），这是一种天然存在的细胞毒性物质。它们的生化活性是去纯化rRNA普遍保守的sarcin/ricin loop (SRL)，导致被感染细胞停止蛋白质合成。美洲商陆抗病毒蛋白（PAP）是一种具有广谱抗病毒活性的I型RIP蛋白。除了典型的rRNA去嘌呤活性外，PAP还具有与其他rip不同的mRNA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，并且是破坏真核生物翻译的代理的模型。这个项目的目标是表征机制，通过PAP选择RNA目标去纯化。PI将采用生物物理和生化方法的结合来(1)确定真核起始因子（eIF）参与RNA选择的程度和亲和力；(2)确定影响PAP亲和力和酶活性的基本mRNA结构元件。具有不同二级结构的带帽和未带帽mRNA的寡核苷酸将在本研究中用于阐明这些结构要求。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。这种对植物防御机制的了解将指导减少病原体感染造成的作物损失的实际策略。更广泛的影响：这项研究将主要由纽约城市大学约翰杰伊学院的本科理科专业学生进行。纽约市立大学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，培训将在一个拥有超过60%的西班牙裔和非洲裔美国学生的校园进行，这是美国东北部最大的西班牙裔服务机构，也是全国第四大机构。在她在科学系的头五年里，PI在她的研究实验室共培养了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民族，除了两名之外都是女性。这个项目的教育影响包括现代生物物理和生化技术的培训，这些技术是实验室课堂技能的延伸。该研究包括适合不同层次（高中、本科、研究生和研究生）学生参与的方法，从而在培养具有科学素养的公民的同时提供全面的培训。完成培训后，学生将为高级学位课程和/或STEM领域的职业做好充分准备。这项研究在方法上严重依赖于同行作为共同研究人员；因此，合作和团队合作技能自然被纳入学生的训练。除了为本科生提供研究机会外，该项目还允许参与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联系，并与未来的研究生院代表会面。通过这个项目，学生们将了解到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、更健壮的农作物、安全的粮食供应保护和国家经济。

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.