UNDERSTANDING AND MANIPULATING RNA SILENCING

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0215716
• Project No.: CA-R-BPS-7758-H
• Project Start Date: Mar 1, 2008
• Project End Date: Feb 28, 2013

Project Director
chen, X.

Recipient Organization
UNIVERSITY OF CALIFORNIA, RIVERSIDE
(N/A)
RIVERSIDE,CA 92521

Performing Department
BOTANY AND PLANT SCIENCES

Non Technical Summary
Small RNAs (short strands of ribonucleic acids, a type of molecule bearing genetic information) have recently emerged as key regulators in a wide array of biological processes in both plants and animals, such as developmental patterning, response to environmental stresses such as drought, heat, cold or flooding, and defense against bacterial and viral pathogens. In this project, we wish to uncover the mechanisms underlying the synthesis, degradation, and function of small RNAs. We will use classical genetic approaches to identify proteins that mediate the accumulation or function of small RNAs. In addition, in order to harness the power of small RNAs to enhance plants¿ defense against pathogens, we also wish to isolate chemicals that enhance the accumulation or potency of small RNAs. Such a chemical would be widely applicable to crop species to fight against pathogen infection.

Animal Health Component

(N/A)

Research Effort Categories

Basic

50%

Applied

(N/A)

Developmental

50%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	2420	1080	20%
202	2420	1080	20%
203	2420	1080	20%
206	2420	1080	20%
212	2420	1080	20%

Knowledge Area
202 - Plant Genetic Resources; 203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 212 - Pathogens and Nematodes Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms; 206 - Basic Plant Biology;

Subject Of Investigation
2420 - Noncrop plant research;

Field Of Science
1080 - Genetics;

Keywords

microrna

small interfering rna

rna silencing

exonuclease

uridylation

viral pathogen

bacterial pathogen

chemical

Goals / Objectives
Our long-term goals are to uncover the molecular mechanisms underlying RNA silencing and to manipulate the process to improve crop yield/quality and to increase the tolerance of plants for biotic and abiotic stresses. RNA silencing is a broad term that refers to newly discovered processes whereby small RNAs of 21-24 nucleotides repress target gene expression in a sequence-specific manner. RNA silencing is crucial for a broad array of biological processes including developmental patterning, maintenance of genome stability, response to abiotic stresses, and defense against bacterial and viral pathogens. Understanding the biogenesis, turnover, and functions of these small RNAs will ultimately allow us to employ small RNA-based technology to improve agriculture and natural resources. Furthermore, transgenic technology, which holds the promise for rapid crop improvements in the future, is universally affected by RNA silencing. Transgenes, which are sensed by plants as foreign DNA, are under the attack of RNA silencing so that transgene expression may not be stable over generations. Understanding the mechanisms of RNA silencing will allow us to manipulate plants to avoid transgene silencing. Objectives We wish to address the two main outstanding questions in miRNA biology in the next five years with a combination of forward genetics, reverse genetics, and chemical genetics, as well as biochemical approaches. The objectives are: 1) Identify genes that act negatively in miRNA biogenesis or function 2) Isolate chemicals that affect the potency of miRNA-mediated regulation Outputs 1. Isolation of chemicals that affect the potency of small RNA-based regulation. A chemical that enhances the function of small RNAs can be used to protect crops when there is a viral or bacterial infection. Since plants normally use RNA silencing to fight off bacterial and viral infection, boosting this natural response of plants with a chemical when they are infected is a readily applicable way to control diseases in a variety of crops. The advantages include the applicability to all crop species, the broad-spectrum effect against most if not all pathogens, and the timed usage (only when there is an infection). 2. Understanding how small RNAs are synthesized and how small RNAs regulate their target genes will enable us to efficiently design small RNA-based technology to engineer drought-, heat-, cold-tolerance and disease resistance in crop plants. For example, artificial miRNAs can be made to repress any target gene of interest in a sequence-specific manner.

Project Methods
We will take reverse genetics, forward genetics, and chemical genetics approaches to the identification of genes or chemicals that negatively impact miRNA biogenesis or function. Aim 1. Identify genes that act negatively in miRNA biogenesis or function 1A. A reverse genetics approach to the identification of enzymes that turnover miRNAs 1B. A forward genetics approach to the identification of genes that negatively impact miRNA metabolism For aim 1A, we will obtain T-DNA mutants (from the Arabidopsis Stock Center) in selected genes that potentially encode the enzymatic activities that we are looking for. We will confirm that the mutants are indeed affected in the specific genes. Then we will analyze these mutants for the levels or the uridylation status of miRNAs. If these genes are confirmed to play a role in miRNA biogenesis, we will perform biochemical assays on one or two of the proteins to elucidate their biochemical activities. For aim 1B, we will randomly mutagenize wild type plants and search for mutations that lead to increased activities of miRNAs (i.e. lower levels of miRNA targets). These mutatns will lead us to genes that act negatively in miRNA biogenesis or function. A luciferase gene containing a miRNA target site would be a very sensitive reporter of the level or activity of the miRNA. Live imaging can be easily applied to the plant to screen for ones with lower levels of luciferase activity. We have constructed a reporter gene in which a luciferase gene with a miR173-binding site is controlled by the 35S promoter (35S::luc-miR173). We then crossed the transgene into the rdr6-11 background containing a point mutation in RDR6. Since RDR6 is required for sense transgene silencing, the 35S::luc-miR173 transgene will not be silenced in the rdr6 background (silencing of the transgene will be problematic for our screen). We will mutate seeds of rdr6-11 35S::luc-miR173 plants with ethyl methane sulfonate (EMS) and harvest M2 seeds in single families. The M2 seedlings will be imaged (live) for luciferase activity and mutants with lower levels of luciferase activity will be collected as potential mutants. The mutants will be further analyzed and the corresponding genes will be cloned. Aim 2. Isolate chemicals that affect the potency of miRNA-mediated regulation We will screen various chemical libraries on seedlings of rdr6-11 35S::luc-miR173 to identify chemicals that decrease the luciferase activity of the seedlings. Such chemicals likely lead to increased accumulation or activity of miRNAs. Seeds will be germinated in solutions containing the chemicals in a 96-well format. The seedlings will be washed and imaged for luciferase levels. Once chemicals that cause lower levels of luciferase are identified, a secondary screen will be performed. We will examine the profiles of small RNAs to determine whether the chemicals affect the levels of miRNAs. If a chemical leads to increased accumulation of miRNAs, it likely promotes miRNA biogenesis. On the other hand, if a chemical leads to reduced miRNA target gene expression without affecting miRNA levels, it likely promotes miRNA function.

Progress 03/01/08 to 02/28/13

Outputs
OUTPUTS: Activities: The objectives of the project (7758-H) are: 1) to identify genes that affect the biogenesis or stability of small RNAs and 2) to search for chemicals that affect the efficiency of RNAi-based regulatory systems. We have conducted extensive experimentation towards, and achieved, both objectives. For objective 1, we have identified two new genes, DAWDLE (DDL) and SDN1, which act in the biogenesis and stability of small RNAs, respectively. For objective 2, we have screened chemical libraries available at UCR and identified two chemicals that release gene silencing. Teaching: The PI trained one graduate student (Theresa Dinh) and six undergraduate students in this project. Theresa Dinh performed the chemical screen described above and is currently following up on the chemicals that she has identified. This work will be part of her Ph.D. thesis. One of the undergraduate students, Michael O'Leary, worked with Theresa on the chemical screen. This research experience solidified his determination to pursue a career in agricultural science. He applied to graduate school in the fall of 2008 and has been interviewed at UC Davis, UC San Diego, and Cornell. Mentoring: The PI has mentored a postdoctoral fellow (Bin Yu) and a research specialist (Vanitharani Ramachandran) in this project. The PI had weekly meetings with the two researchers and guided them to write manuscripts, to give research presentations at international meetings, and to interview for jobs. Bin Yu moved on to become an assistant professor at University of Nebraska, Lincoln. Events: The PI presented the work at the following conferences or seminars. 1. Seminar Speaker at UC Davis, Jan 16, 2009. 2. Invited speaker at Ray Wu Memorial Symposium, Cornell University, Oct 3-4, 2008. 3. Invited speaker at Epigenome Conference, Blue Mountains, Australia, Sept. 25-28, 2008 4. Invited speaker at Ray Wu symposium, Beijing University, China, July 10, 2008 5. Invited speaker at 6th Canadian Plant Genomics Workshop, Toronto, Ontario, Canada. June 23-26, 2008. 6. Invited speaker at 3rd Microsymposium on Small RNAs, Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria. May 19-21, 2008. 7. Seminar speaker at Okalahoma State University, Stillwater, Okalahoma. April 4, 2008. 8. Seminar speaker at Texas Tech University, Lubbock, Texas. April 2, 2008; 9. Seminar speaker at University of Arizona, Tucson, Arizona. Jan 22, 2008. Vanitharani Ramachandran was an invited speaker in the Small RNA workshop at the Plant and Animal Genome meeting, San Diego, CA, Jan 10, 2009. Dissemination:Our research findings were disseminated by publications and the above-mentioned oral presentations at conferences. PARTICIPANTS: PI: Xuemei Chen; was responsible for project design and supervision of personnel conducting research Research Specialist: Vanitharani Ramachandran; was responsible for the execution of the SDN1 project. She also trained three undergraduate students. Postdoctoral fellow: Bin Yu; was responsible for the execution of the DDL project. He also trained two undergraduate students. Graduate student: Theresa Dinh; was responsible for the chemical screen (second objective). She also trained one graduate student. Undergraduate students: Michael O'Leary (NSF REU and research for credit); Min Kong (research for credit); Janet Mojica (work study); Lara Do (research for credit); Kelsey Nguyen (research for credit); Kelley Steward (research volunteer); these students participated in various parts of the project under the supervision of the research specialist, the postdoctoral fellow, the student, and the PI. Collaborators: Scott Poethig (University of Pennsylvania) Vicki Vance (University of South Carolina) Blake C. Meyers (University of Delaware); Xiaofeng Cao (Institute of Genetics and Developmental Biology, China) David Chevalier (Missisipi State University) Thierry Lagrange (Universite de Perpignan, France); John C Walker (University of Missouri) The project provided a training opportunity for the participants in the Chen lab and the undergraduate students involved. Vanitharani Ramachandran published a research paper in Science and was invited to present her work at an international conference. Bin Yu published a first author PNAS paper, which together with publications from other projects, helped him secure an assistant professor position at University of Nebraska, Lincoln. Theresa Dinh, a graduate student has made significant progress towards her Ph.D. degree. She is particularly active in outreach activities such as volunteering in local high schools. Among the six undergraduate students, Michael O'Leary is applying for graduate school, and Kelsey Nguyen had a Dean's fellowship for research in the summer TARGET AUDIENCES: Outreach: the target audiences for outreach are 1) high school students; 2) undergraduate students who are unsure about their future careers; 3) graduate students and postdoctoral fellows who have decided to pursue careers in sciences and education Theresa Dinh volunteers at local high schools and attends meetings of undergraduate women's groups to share her experience of what it takes to be a scientist. We host many undergraduate students in the lab to provide research experience for them. For graduate students and postdocs, weekly lab meetings, journal clubs and one-on-one meetings with the PI help prepare them to achieve their career goals. Research: The target audiences of our research findings are scientists in academia and industry. Our findings will guide the design of new therapeutic agents in treating human diseases. A preliminary disclosure on the SDN1 enzyme has been filed. UCR is deciding whether to proceed with a patent application. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Our work in this project resulted in a change in knowledge (high impact publications in journals such as Science, PNAS, Dev Cell etc). We found that DDL in Arabidopsis and its homolog SNIP1 in humans act in microRNA biogenesis. We also identified the first class of exonucleases (SDN1 and its homologs) that degrade mature microRNAs in Arabidopsis. Homologs of SDN1 are found in animals including humans. Our work also showed that the turnover of small RNAs is important in their homeostasis - reduction of SDN1 function results in increased microRNA levels in vivo and consequently pleiotropic developmental defects. The SDN1 enzyme is potentially important in evaluating the stability of small RNAs that are to be used in medical therapy. Small RNAs are currently being developed as agents in medical treatments. The in vivo stability of exogenously introduced small RNAs is a great issue hampering the use of small RNAs as treatment agents. Since SDN1 is one of the enzymes that degrade small RNA in vivo, it can be used to evaluate the stability of small RNAs in vitro and guide the design of modified small RNAs that are more stable in vivo. A preliminary disclosure of this discovery has been processed. UCR will decide whether it is going to proceed to patent application.

Publications

• 1.Blake C. Meyersa, Michael J. Axtell, Bonnie Bartel, David P. Bartel, David Baulcombe, John L. Bowman, Xiaofeng Cao, James C. Carrington, Xuemei Chen, Pamela J. Green, Sam Griffiths-Jones, Steven E. Jacobsen, Allison C. Mallory, Robert A. Martienssen, R. Scott Poethig, Yijun Qi, Herve Vaucheret, Olivier Voinnet, Yuichiro Watanabe, Detlef Weigel and Jian-Kang Zhu. (2008). Criteria for Annotation of Plant MicroRNAs. Plant Cell 20, 3186-3190.
• 2. Vanitharani Ramachandran and Xuemei Chen*. (2008). Degradation of microRNAs by a family of exoribonucleases in Arabidopsis. Science 321, 1490-1492.
• 3. Bin Yu, Liu Bi, Binglian Zheng, Lijuan Ji, David Chevalier, Manu Agarwal, Vanitharani Ramachandran, Wanxiang Li, Thierry Lagrange, John C Walker, and Xuemei Chen*. (2008). The FHA domain proteins DAWDLE in Arabidopsis and SNIP1 in humans act in small RNA biogenesis. PNAS 105, 10073-10078.
• 4. Vanitharani Ramachandran and Xuemei Chen*. (2008). Small RNA metabolism in Arabidopsis. Trends in Plant Science 13, 368-374.
• 5. Xuemei Chen*. (2008). A silencing safeguard: links between RNA silencing and mRNA processing in Arabidopsis. Dev. Cell 14, 811-812.
• 6. Jixian Zhai, Jun Liu, Bin Liu, Pingchuan Li, Blake C. Meyers, Xuemei Chen and Xiaofeng Cao*. (2008). Small RNA-directed epigenetic natural variation in Arabidopsis thaliana. Plos Genetics 4(4):e1000056.
• 7. Sizolwenkosi Mlotshwa, Gail J. Pruss, Angela Peragine, Matthew W. Endres, Junjie Li, Xuemei Chen, R. Scott Poethig, Lewis H. Bowman, and Vicki Vance (2008). DICER-LIKE2 plays a primary role in transitive silencing of transgenes in Arabidopsis. Plos One 3(3), e1755.
• 8. Julien Curaba and Xuemei Chen. (2008). Biochemical activities of Arabidopsis RNA-dependent RNA polymerase 6. Journal of Biological Chemistry 283, 3059-3066.
• 9. Xuemei Chen. (2008). MicroRNA metabolism in plants. Chapter 6 in RNA interference, Current Topics in Microbiology and Immunology, edited by P. Paddison and P. K. Vogt, published by Springer-Verlag Berlin Heidleberg.