ROLES OF MICRORNA STRUCTURES IN PLANT RNA SILENCING

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0213316
• Project Start Date: Oct 1, 2007
• Project End Date: Sep 30, 2011
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF KENTUCKY
500 S LIMESTONE 109 KINKEAD HALL
LEXINGTON,KY 40526-0001

Performing Department
Plant and Soil Sciences

Non Technical Summary
microRNAs (miRNAs) are recently discovered endogenous small RNAs of ~22 nucleotides in plants and animals. They regulate gene expression after the gene transcription by cleaving the mRNA and prevent mRNA from translation into proteins. Various miRNAs from both plants and animals were cloned and their precursors and genes were identified. Stem-loop RNA structure is the hallmark of miRNA precursors and helps produce highly efficient RNA silencing triggers, the miRNAs. Here we propose to dissect the miRNA precursor structures using informatics approach and find rules of their structures in RNA silencing. This will involve the establishment of a miRNA structure database and its analysis in detail. The rules found from the database analysis will be applied to design highly efficient small RNAs or small RNA vectors for silencing any genes of interest. The proposed activities also include a training of three graduate students and several undergraduate students.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	2499	1040	100%

Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
2499 - Plant research, general;

Field Of Science
1040 - Molecular biology;

Keywords

microrna (mirna)

rnai

sirna

rna silencing

microrna vector

database

bioinformatics

rna secondary structures

Goals / Objectives
Objectives: 1. To generate a miRNA duplex database (miRDubase) and analyze the records in the database for miRNA duplex rules using a bioinformatics approach; 2. To determine the mechanisms and applications of the miRNA-like siRNA duplexes or premiRNA-like structures in RNAi in vitro and in vivo. Outputs: 1. We expect that our proposed bioinformatics analysis will result in a new miRDubase, comprehensive rules of miRNA duplex structures, a framework of miRNA-like siRNA designing software, and a web-based online service for miRNA-like siRNA, or vector designing; 2. The expected outcomes from this aim will be the generation of experimentally tested and optimized rules for miRNA-like siRNA duplex designing. We expect to uncover the mechanisms by which an efficient RISC assembly and target cleavage are fulfilled. We also expect to revolutionize the current siRNA design with new rules and develop new miRNA-like vectors for gene silencing in plants and animals. These expected rules will be published in journals and on a website for public access.

Project Methods
1.2.1. Establishing a new miRDubase. Methods: The raw data will be taken directly from the public miRBase. In order to transform the miRBase into the miRDubase, all the pre-miRNA duplexes from specific plants will be conceptually diced to generate miRNA duplexes. 1.2.2. Analyzing the miRDubase. Methods. The analysis of the miRNA duplexes from the miRDubase will include the following parameters: (1) duplex length distribution; (2) mismatch distribution; (3) bulge distribution; and (4) AU/UA and CG/GC distribution. In our preliminary study, these analyses were conducted using Excel analysis. Here, we will design a computer program (for details, see below) to carry out these analyses in a large-scale manner. 1.2.3. Generation of comprehensive miRNA duplex rules for programming and the web-based interface service for customer designing of miRNA-like siRNAs or vectors. Methods: After we generate the miRNA duplex length, mismatch, bulge, and AU/UA & CG/GC distributions using the above-mentioned method, we will plot the distributions along the duplex from position 1 to position 19 (if the average miRNA size is 21 nt). The peak position of the mismatches, bulges, and AU/UA or CG/GC will be understood as the general rules for designing miRNA-like siRNAs. 1.2.4. Computer programming technology. Methods: The computer technology infrastructure will be comprised of a Structured Query Language (SQL) server, a web server, and a customized reporting system. The servers and reporting system will be based on Microsoft system. The computer language platform to support this infrastructure will be a combination of SQL and Perl and/or Java. 2.2.1. To test the in vitro RISC assembly with miRNA-like siRNA duplexes that have one to four mismatches and two open ended structures. Methods: The design of an miRNA-like siRNA duplex will follow the methods established in our preliminary experiments. The control experiments will be set using traditional siRNA with perfect duplex structures. After chemical synthesis of the two strands following the above rules, the methods for duplex annealing, RISC assembly, and target cleavage will follow our published original or method papers. 2.2.2. To test, in vitro, the single-stranded pre-miRNA-like RNA structures in RISC assembly and subsequent target RNA cleavage. Methods: The design for pre-miRNA-like structures will further experimentally explore the structural rules found in our preliminary bioinformatics analysis. 2.2.3. To test, in vivo, miRNA-like siRNA duplexes or the single-stranded pre-miRNA-like RNA structures in gene silencing in cells. Methods: In vitro RNAi triggered by miRNA-like siRNA duplexes or single-stranded pre-miRNA-like structures will be further tested in vivo in cells. We will use plant cells (protoplasts) corresponding to the in vitro experiments. 2.2.4. To improve online service program for better designing of miRNA-based siRNA duplexes or vectors. Methods: Based on a series of in vitro experimental results, we will be able to improve our web-based online service by providing various choices. All the choices will be made possible through an online interface.

Progress 10/01/07 to 09/30/11

Outputs
OUTPUTS: Investigator has left the university PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Investigator has left the university

Publications

• No publications reported this period

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: We created an online database to demonstrate the microRNA structures and compositions. This database can be reached online to users. The database was also used for teaching and mentoring graduate students. We attended various symposia and workshops to demonstrate the basic research results and applications We provided consulting, counseling, and tutoring to collaborators and colleagues inside and outside this university. We created the mirnainfo database (http://mirnainfo.com/miRNAResearchHome.aspx) We provided service to researchers inside and outside this university regarding how to design effective artificial miRNAs based on the outcomes of this project. We also created a teaching website (http://www.uky.edu/%7Egtang2/Teaching.html) for the education of graduate and undergraduate students. This educational website can be reached to users from inside and outside this university. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
RNAi and microRNA added cutting-edge knowledge to the education of the public and the academic. The outcome of this project led to several research and education publications. We generated improved artificial miRNA vector that can express single or multiple endogenous miRNA or artificial miRNAs to target any gene for silencing. This vector was used to over express the endogenous miR165/166 to study the role of miR165/166 in flower development in collaboration with Dr. Xuemei Chen at UC Riverside.

Publications

• Xiaoyun Jia, Venugopal Mendu, and Guiliang Tang (2010) An array platform for identification of stress-responsive miRNAs in plants. Methods Mol Biol. 639: 253-69.
• Guiliang Tang (2010) Plant microRNAs: An insight into their gene structures and evolution. Seminars in Cell and Developmental Biology 21:782-789.(PDF)
• Dianwei Han, Jun Zhang, and Guiliang Tang (2011) MicroRNAfold: pre-microRNA secondary structure prediction based on Modified NCM model with thermodynamics-based scoring strategy. International Journal of Data Mining and Bioinformatics (In press)
• Xiaoyun Jia, Jun Yan, and Guiliang Tang (2011) MicroRNA-mediated DNA methylation in plants. Frontiers in Biology (In press)
• Lijuan Ji, Xigang Liu, Jun Yan, Wenming Wang, Rae Eden Yumul, Yu Ju Kim, Thanh Theresa Dinh, Jun Liu, Xia Cui, Binglian Zheng, Manu Agarwal, Chunyan Liu, Xiaofeng Cao, Guiliang Tang, and Xuemei Chen* (2011) ARGONAUTE10 and ARGONAUTE1 Regulate the Termination of Floral Stem Cells through Two microRNAs in Arabidopsis. PLOS Genetics (In press)

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: We have completed the analysis of pre-miRNA structures and generated practical rules for plant RNA silencing using modified two-hit artificial miRNAs (amiRNAs) to silence any genes of interest. The silencing efficiency reached to 90% compared to tradition amiRNA technology. We are now applying this technology to silence families of transcription factor genes in Arabidopsis. Translational application to crops will be conducted in the future for crop improvement. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Plant researchers and crop breeders. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Outcomes: 1. Novel two-hit amiRNA vector for gene silencing in plants; 2. Functional genomics for plant transcription factor genes. Impact: 1. Tools for plant gene functions; 2. Crop improvement.

Publications

• Xiaoyun Jia, Wang-Xia Wang, Ligang Ren, Qi-Jun Chen, Venugopal Mendu, Benjamin Willcut, Randy Dinkins, Xiaoqing Tang, and Guiliang Tang (2009) Differential and dynamic regulation of miR398 and its targets in response to ABA and salt stress in Populus tremula and Arabidopsis thaliana. Plant Molecular Biology 71: 51-59.
• Xiaoyun Jia, Ligang Ren, Qi-Jun Chen, Runzhi Li and Guiliang Tang (2009) UV-B responsive microRNAs in Populus tremula. J. of Plant Physiology 166: 2046-2057.
• Ricky Lewis, Venugopal Mendu, David McNear, and Guiliang Tang (2009) Roles of microRNAs in Plant Abiotic Stress, Molecular Techniques in Crop Improvement. 2nd Edition, edited by S. Mohan Jain and D.S. Brar. Springer Netherlands, pp 357-372.
• Mian Gu, Ke Xu, Aiqun Chen, Yiyong Zhu, Guiliang Tang, and Guohua Xu (2010) Expression Analysis Suggests Potential Roles of microRNAs for Phosphate and Arbuscular Mycorrhizal Signaling in Solanum lycopersicum. Physiol Plant.138(2):226-37.
• Xiaoyun Jia, Venugopal Mendu, and Guiliang Tang (2010) An array platform for identification of stress-responsive miRNAs in plants, "Methods in Molecular Biology; Plant Stress Tolerance- Methods and Protocols" edited by Ramanjulu Sunkar, Oklohoma State University, Stillwater, OK, USA. HUMANA PRESS.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: This project is still going on. We have generated many useful data from experimental testing. Hopefully, we will generate more concrete rules regarding the role of miRNA secondary structure in gene silencing. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We generated online miRDuBase and miRNA designer.

Publications

• Peter T. Nelson, Wang-Xia Wang, Bernard R. Wilfred, Guiliang Tang (2008) Technical variables in high-throughput miRNA expression profiling: Much work remains to be done. BBA-Gene Regulatory Mechanisms 1779: 758-765
• Guiliang Tang, Yu Xiang, Zhensheng Kang,Venugopal Mendu, Xiaohu Tang, Xiaoyun Jia, Qi-Jun Chen, and Xiaoqing Tang (2008) Small RNA technologies: siRNA, miRNA, antagomiR, target mimicry, miRNA sponge and miRNA profiling. Current Perspectives in MicroRNAs, Springer Netherlands, pp 17-33
• Guiliang Tang, Xiaoqing Tang, Venugopal Mendu, Xiaohu Tang, Xiaoyun Jia, Qi-Jun Chen, and Lihen He (2008) The art of microRNA: various strategies leading to gene silencing via an ancient pathway. BBA-Gene Regulatory Mechanisms 1779: 655-662
• Guiliang Tang (2008) MicroRNAs: An exciting and open field calls for extensive study from initial and established investigators. BBA-Gene Regulatory Mechanisms 1779: 653-654