Progress 09/01/11 to 08/31/13
Outputs
Target Audience: The target audiences of this project are basic researchers interested in understanding plant and viral translation mechanisms, the structure and function of viral RNAs and their interaction with host proteins. Additionally, this project also seeks to delineate the basic rules of RNA and RNA-protein structure that could be of interest to biochemistry students. Changes/Problems: As indicated in the annual progress report the project was modified to take into account a recent study by Wang et al., 2011. Structure 19, 868-880 in which a modeled tertiary structure of Panicum mosaic virus-like cap-independent Translation Element (PMV PTE) alone and bound to translation initiation factor eIF4E was featured. The PD’s grant also proposed to determine tertiary structure of the PMV PTE by X-ray crystallography. Given the fact that PTE elements from the other viral genera in this study were all predicted to adopt a similar tertiary structure and that structure is more conserved than sequence, it seems that X-ray crystallographic PTE structure determination would not yield new insights into plant virus translation mechanisms. Thus, this new information resulted in a shift in project focus to conducting mutagenesis, translation assays and structure probing to validate this structure and further understand the molecular mechanism of the PTE mediated cap-independent translation initiation. What opportunities for training and professional development has the project provided? Training activities This project allowed for the training of visiting and junior scientists in RNA structure analysis, improving PD’s teaching skills, and increasing the impact of the project. Specifically, PD trained Iowa State University MS students Mariko S. Peterson and Sannie J. Olson in basic biochemical techniques such as RNA and protein synthesis and purification, cloning, gel electrophoresis and polymerase chain reaction. Also, PD provided technical advice on solution structure probing to Dr. Natalia N. Singh, Adjunct Assistant Professor of Biomedical Sciences at Iowa State University. Training opportunities provided by PD Fellowship Grant extent beyond Iowa State University to include Ph.D students training in viral RNA structure analysis Sohoni Das Sharma, Chemistry Department at Hunter Collage, City University of New York, and Manuel Miras Marin, Centro de Edafología y Biología Aplicada del Segura (CEBAS), Consejo Superior de Investigaciones Científicas (CSIC), Spain, which resulted in a submitted manuscript to PLoS Pathogens. Professional development PD’s Fellowship Grant allowed opportunities to share project accomplishments and increase knowledge of new experimental approaches to study RNA structural properties at local, national and international scientific meetings. Also, Dr. Kraft participated in a Center for Integrating Research, Teaching and Learning (CIRTL) course: Teaching-as-Research in Science, Technology, Engineering and Mathematics (STEM) Courses and is accepted into a highly competitive professional development program entitled Preparing Future Faculty at Iowa State University this fall to further improve PD’s instructing abilities by enhancing teaching and online course delivery methods. How have the results been disseminated to communities of interest? The project results were presented at the 2011 EMBO Conference Series (EMBL Heidelberg, Germany), RNA 2012: The 17th Annual Meeting of the RNA Society (Ann Arbor, Michigan), 2012 AFRI NIFA Fellows Project Directors Meeting and in a talk at Iowa State University Department of Biochemistry. Further more, Dr. Kraft shared the obtained results and expertise in RNA structural analysis by providing mentoring and consulting services to scientific community on and off Iowa State University campus, resulting in one published and one submitted journal article. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1) Major activities completed: Toward these goals, untranslated regions of nine different viruses, including additional coding sequences of certain viruses in the Carmovirus, Panicovirus and Umbravirus genera were cloned into a luciferase plasmid downstream of a T7 phage promoter. mRNAs transcribed from these plasmids were added to a wheat germ translation extract, a cell-free translation system derived from tobacco Bright Yellow-2 (BY-2) cells and oat protoplast followed by luciferase measurements. The two in vitro translation systems were also used to test PTEs from viruses belonging to three viral genera for their ability to inhibit translation of reporter mRNAs in trans-inhibition assays. Mixing a reporter mRNA along with 100-fold excess of the PTE from each virus to serve as an inhibitor of the translation of the mRNA shows how well the PTE can compete for, bind, and sequester the factors of interest. The PTE dynamic structural properties belonging to three different viral genera were investigated under various ionic conditions and the key panicovirus nucleotides hypothesize to bind into cap-binding pocket of eIF4E were mutated and their functional and structural properties investigated. The investigator provided both consulting and tutoring services to graduate students from Hunter College, New York, Iowa State University and Spain. 2) Specific objectives met: High-resolution mapping and structural analysis of sequences required for PTE mediated cap-independent translation was conducted to reveal the presence of stable fold at physiological magnesium ion concentrations in both Umbravirus and Panicovirus genera. In contrast, Carmovirus PTEs adopt a stable fold that is independent of magnesium. This fold is further stabilized via a pseudoknot interaction within the Panico and Umbravirus PTEs, which interacts with the key cap-binding translation initiation factor, eIF4E. The secondary structures of diverse PTEs consist of three helices radiating from a central hub and they all harbor a conserved G residue hypothesized to insert into cap-binging pocket of eIF4E. Mutagenesis of this conserved residue in Panicoviruses revealed that replacing G with a T residue is tolerated both in vitro and in vivo suggesting that this compact base is not disturbing the cap binding interactions in eIF4E. Experiments comparing efficiencies of PTEs in three different genera and with other translation enhancers revealed that the panicovirus PTE is one of the most efficient cap-independent translation element in plant systems that also works in animal systems. The fact that PTEs fold into defined structures even in the absence of cation bodes well for obtaining meaningful crystal structure goal which has not been met yet. 3) Significant results achieved, including major findings, developments, or conclusions: As a result of provided mentoring and consulting a novel cap-independent translation element (CITE) essential for translation control in Melon necrotic spot virus resistant plants belonging to a new class called the CXTE (CABYV-Xinjiang-like translation element) was structurally characterized. The CXTE folded into two helices protruding from a central hub in magnesium independent manner and is able to control translation in the absence of eIF4E protein. Another result of student mentoring is identification of 80S ribosome pausing at the start codon on barley yellow dwarf virus reporter mRNA suggesting mechanism entrance via the 5’ end and not the through mediation of CITE from the 3’ end. Also, structural studies on the Barley yellow dwarf virus-like translation elements (BTE) revealed that Mg2+ stabilizes BTEs of all three types by charge neutralization and not by coordination to a specific site(s), and that the BTEs fold into defined, functional structures in the absence of protein that is unchanged by eIF4G binding. This supports the model in which the BTE base pairs simultaneously to the 5’ UTR and the eIF4F complex to allow the 40S ribosomal subunit recruitment to the 5’ end. All of these finding are now published with acknowledgement of NIFA funding. 4) Key outcomes or other accomplishments realized: All plant viruses are parasites of the cell’s protein synthesis machinery (ribosomes). Viruses use non-canonical translation (protein synthesis) mechanisms to induce the ribosomes to synthesize viral proteins encoded in the viral mRNA. Notably, nine viruses in the Carmovirus, Panicovirus and Umbravirus genera were all found to translate highly efficiently in plant cell extracts and in plant protoplast. In particular, Panicovirus reporter mRNAs translated 2-8 fold (depending on a virus used) higher than either Umbra or Carmovirus reporter mRNAs in vivo (cells). Further investigation revealed that these viruses utilize a powerful and highly structured 100-nucleotide Panicum mosaic virus-like translation element (PTE). Analysis of the PTE dynamic structural properties suggests the presence of stable fold at physiological magnesium ion concentrations. This RNA fold is further stabilized via a structure called a pseudoknot within the PTE, which in turn, enables the key interaction with the cap-binding translation initiation factor, eIF4E. However, magnesium dependent pseudoknot was observed only in Panicovirus and Umbravirus and not in the Carmovirus PTEs suggesting that Carmovirus RNAs utilize an alternative folding and mechanism of initiation machinery recruitment. These data enhance understanding of the mechanism of cap-independent, and possibly, by comparison, cap-dependent translation initiation. Further more, the data warrant further characterization of this remarkable ability of the PTE to bind eIF4E in the absence of a 5’ cap that could lead to new ways to limit plant virus infection. Also the information from this research will further understanding of translation in general and may lead to better means of controlling gene expression in crop plants and other higher organism.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Kraft, J. J., Treder, K., Peterson, M., and W. A. Milller. 2013. Cation-dependent folding of 3' cap-independent translation elements facilitates interaction of a 17 nucleotide conserved sequence with eIF4G. Nucleic Acids Research 41, 3398-3413.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2013
Citation:
Miras, M., Sempere, R. N., Kraft, J. J., Miller, W. A., Aranda, M. A., and V. Truniger. 2013. Interfamilial recombination between viruses led to acquisition of a novel translation enhancing RNA element that extends viral host range. Manuscript submitted to PLoS Pathogens.
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Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: Activities: The goal of this project is to understand the mechanism of messenger RNAs of certain plant viruses, which contain protein synthesis (translation) enhancer sequences called Panicum mosaic virus-like elements (PTEs). Toward this goal, untranslated regions of nine different viruses were cloned into a luciferase plasmid downstream of a T7 phage promoter. mRNAs transcribed from these plasmids were added to a wheat germ translation extract or a cell-free translation system derived from tobacco Bright Yellow-2 (BY-2) cells followed by luciferase measurements. The two translation systems were also used to test PTEs from viruses belonging to three viral genera for their ability to inhibit translation of reporter mRNAs in trans-inhibition assays. Mixing a reporter mRNA along with 100-fold excess of the PTE from each virus to serve as an inhibitor of the translation of the mRNA shows how well the PTE can compete for, bind, and sequester the factors of interest. The PTE dynamic structural properties are now being investigated under various ionic conditions. Events and Services: The project accomplishments were shared with local, national and international scientific community at several meetings. Specifically, project results were presented at 2011 EMBO Conference Series (EMBL Heidelberg, Germany), RNA 2012: The 17th Annual Meeting of the RNA Society (Ann Arbor, Michigan), 2012 AFRI NIFA Fellows Project Directors Meeting and in a talk at Iowa State University Department of Biochemistry. Both consulting and tutoring services were provided by the investigator. A graduate student from Hunter College, New York, and a graduate student from Spain were trained by Dr. Kraft. PARTICIPANTS: Dr. Jelena J. Kraft, Postdoctoral Research Associate at Iowa State University is the Fellowship Grant PD. Dr. Kraft designed and performed proposed experiments. Also, the PD collected and analyzed data and shared it with national and international scientific community at annual meetings. Dr. W. Allen Miller, Professor of Plant Pathology and Microbiology and Director of Center for Plant Responses to Environmental Stresses at Iowa State University served as the Fellowship Grant PD, Jelena Kraft's mentor. As a well-established investigator in the area of plant virus translation mechanisms, Dr. Miller consulted PD on the project objectives on which to focus and on data interpretation. Further more, Dr. Miller provided PD with the materials and most of the equipment necessary to perform the research. Dr. Miller was instrumental in improving PD's writing skills by encouraging participation in proposal writing and grant writing workshops. Collaborators and contacts: PD continued collaborations with experts in structural biology, Dr. Jeffrey S. Kieft at University of Colorado, School of Medicine, HHMI, and Dr. Julie A. Hoy, Macromolecular Crystallography Facility manager at the California Institute of Technology who provided advice on structural aspects of PD grant. Training and professional development: PD's Fellowship Grant served as a platform to train visiting and junior scientists in RNA structure analysis, improving PD's teaching skills, and increasing the impact of the project. Specifically, PD trained Iowa State University MS student Mariko S. Peterson in basic biochemical techniques such as RNA and protein synthesis and purification, cloning, gel electrophoresis and polymerase chain reaction. Also, PD provided technical advice on solution structure probing to Dr. Natalia N. Singh, Adjunct Assistant Professor of Biomedical Sciences at Iowa State University. Training opportunities provided by PD Fellowship Grant extent beyond Iowa State University to include Ph.D students training in viral RNA structure analysis Sohoni Das Sharma, Chemistry Department at Hunter Collage, City University of New York and Manuel Miras Marin, Centro de Edafologia y Biologia Aplicada del Segura (CEBAS), Consejo Superior de Investigaciones Cientificas (CSIC), Spain. TARGET AUDIENCES: The target audiences of this project are basic researchers interested in understanding plant and viral translation mechanisms, structure and function of viral RNAs and their interaction with host proteins. Additionally, this project also seeks to delineate the basic rules of RNA and RNA-protein structure that could be of interest to biochemistry students. PROJECT MODIFICATIONS: In a recent study by Wang et al., 2011. Structure 19, 868-880, a modeled tertiary structure of Panicum mosaic virus-like cap-independent Translation Element (PMV PTE) alone and bound to translation initiation factor eIF4E was featured. The PD's grant also proposed to determine tertiary structure of the PMV PTE by X-ray crystallography. Given the fact that PTE elements from the other viral genera in this study were all predicted to adopt a similar tertiary structure and that structure is more conserved than sequence, it seems that X-ray crystallographic PTE structure determination would not yield new insights into plant virus translation mechanisms. Thus, this new information resulted in a shift in project focus to conducting mutagenesis, translation assays and structure probing to validate this structure and further understand the molecular mechanism of the PTE mediated cap-independent translation initiation.
Impacts
All plant viruses are parasites of the cell's protein synthesis machinery (ribosomes). Viruses use non-canonical translation (protein synthesis) mechanisms to induce the ribosomes to synthesize viral proteins encoded in the viral mRNA. Notably, nine viruses in the Carmovirus, Panicovirus and Umbravirus genera were all found to translate highly efficiently in plant cell extracts. Further investigation revealed that these viruses utilize a powerful and highly structured 100-nucleotide Panicum mosaic virus-like translation element (PTE). Analysis of the PTE dynamic structural properties suggests the presence of stable fold at physiological magnesium ion concentrations. This RNA fold is further stabilized via a structure called a pseudoknot within the PTE, which in turn, enables the key interaction with the cap-binding translation initiation factor, eIF4E. These data enhance understanding of the mechanism of cap-independent, and possibly, by comparison, cap-dependent translation initiation. Further more, the data warrant further characterization of this remarkable ability of the PTE to bind eIF4E in the absence of a 5' cap that could lead to new ways to limit plant virus infection. Also the information from this research will further understanding of translation in general and may lead to better means of controlling gene expression in crop plants and other higher organisms.
Publications
- No publications reported this period
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