In the past decade, genome-wide profiling of transcripts in plants, animals, and fungi have revealed pervasive transcription in the genome and the presence of multitudes of noncoding transcripts, which do not encode protein products. Increasing evidence points to an essential role of nuclear noncoding RNAs in the regulation of gene expression or genome stability. However, the mechanisms underlying the transcription, processing, and degradation of nuclear noncoding transcripts are almost completely unknown. This project aims to systematically identify nuclear noncoding RNAs by high-throughput sequencing, and to study the mechanisms governing the biogenesis and processing of a subset of such noncoding transcripts, the ones that participate in transcriptional gene silencing to ensure genome stability. The project is part of a long-term effort towards the full understanding of the roles of RNAs in the regulation of genes and genomes.
Broader impacts: This project will illuminate the landscape and biogenesis requirements of plant noncoding RNAs, and thus set the foundation for the scientific community to dissect previously under-appreciated mechanisms of gene and genome regulation. The project will bridge disciplines through collaboration between biologists and computer scientists, train postdoctoral fellows and graduate students in biology to become well versed in bioinformatics, and provide research opportunities in biology to undergraduate students including computer science students.
A revelation of the post-genome era of biology is that genes that encode proteins only constitute a small portion of most eukaryotic genomes. Non-protein-coding RNAs (referred to as noncoding RNAs) are increasingly recognized to be critical players in various biological processes and their malfunction leads to diseases. Although the biological functions of noncoding RNAs are increasingly appreciated, the mechanisms that impact the biogenesis and degradation of these molecules themselves are still poorly understood. The NSF-funded project aimed to uncover the molecular mechanisms that underlie the biogenesis and degradation of noncoding RNAs, with a focus on long noncoding RNAs that are known to mediate gene silencing in the model plant Arabidopsis thaliana. These long noncoding RNAs are crucial for guiding DNA methylation to transposable elements in the genome to ensure genome stability. This model plant is most suitable for the studies because of the strong intellectual foundation for the biology of these long noncoding RNAs and the extensive molecular genetic resources available. In this NSF-funded project, we established two genetic reporter systems that allowed us to effectively identify players involved in the production of a specific class of long noncoding RNAs (those generated by the RNA polymerase Pol V) that act to maintain genome stability by guiding the deposition of DNA methylation. Using these two reporter systems, we conducted chemical and genetic screens and identified chemical compounds and genes that influence RNA-directed DNA methylation. Further studies revealed previously unknown mechanisms in the biogenesis and/or degradation of Pol V-derived long noncoding RNAs. We also uncovered mechanisms that degrade microRNAs, a class of short noncoding RNAs that are important regulators of gene expression. The NSF-funded project helped train postdocs, graduate students, undergraduate students, and a high school student to be skilled in modern biology. In particular, the trainees had opportunities to apply modern genomics technology to their projects and to gain bioinformatics skills to analyze large-scale datasets. These skills are essential in the post-genome era of biology. One postdoc started his own research group and three graduate students obtained their Ph.D. during the funding period. A high school student won many Science Fair awards and went on to college to major in biology. The PI reported results from the NSF-funded research through oral presentations in many countries. The following products were generated in this project. 1. Thanh Theresa Dinh, Michael O’Leary, So Youn Won, Shengben Li, Lorena Arroyo, Xigang Liu, Andrew Defries, Binglian Zheng, Sean R. Cutler, and Xuemei Chen. (2013). Generation of a luciferase-based reporter for CHH and CG DNA methylation in Arabidopsis thaliana. Silence 4:1. 2. Lulu Wang, Xianwei Song, Liangfeng Gu, Xin Li, Shouyun Cao, Chengcai Chu, Xia Cui, Xuemei Chen and Xiaofeng Cao. (2013). NOT2 proteins promote Pol II-dependent transcription and interact with multiple miRNA biogenesis factors in Arabidopsis. Plant Cell 25, 715-727. 3. So Youn Won, Shengben Li, Binglian Zheng, Yuanyuan Zhao, Dongming Li, Xin Zhao, Huilan Yi, Lei Gao, Thanh T Dinh and Xuemei Chen. (2012). Development of a luciferase-based reporter of transcriptional gene silencing that enables bidirectional mutant screening in Arabidopsis thaliana. Silence 2012, 3:6. 4. Tzuu-fen Lee, Sai Guna Ranjan Gurazada, Jixian Zhai, Shengben Li, Stacey A. Simon, Marjori A. Matzke, Xuemei Chen, and Blake C. Meyers. (2012). RNA Polymerase V-dependent small RNAs in Arabidopsis originate from small, intergenic loci including most SINE repeats. Epigenetics 7, 781-795. 5. Guodong Ren, Xuemei Chen and Bin Yu. (2012). Uridylation of miRNAs by HEN1 SUPPRESSOR1 in Arabidopsis. Current Biology 22, 695-700. 6. Yuanyuan Zhao, Yu Yu, Jixian Zhai, Vanitharani Ramachandran, Thanh Theresa Dinh, Blake C. Meyers, Beixin Mo*, and Xuemei Chen. (2012). The Arabidopsis nucleotidyl transferase HESO1 uridylates unmethylated small RNAs to trigger their degradation. Current Biology 22, 689-694. 7. Jun Yan, Yiyou Gu, Xiaoyun Jia, Wenjun Kang, Shanjin Pan, Xiaoqing Tang, Xuemei Chen, and Guiliang Tang. (2012). Effective small RNA destruction by the expression of short tandem target mimic in Arabidopsis thaliana. Plant Cell 24, 415-427. 8. Yun Ju Kim, Binglian Zheng, Yu Yu, So Youn Won, Beixin Mo, and Xuemei Chen. (2011). The role of Mediator in small and long noncoding RNA production in Arabidopsis thaliana. EMBO J. 30, 814-822. 9. Kestrel Rogers and Xuemei Chen. (2013). Biogenesis, turnover and mode of action of plant microRNAs. Plant Cell 25: 2383-2399. 10.Kestrel Rogers and Xuemei Chen. (2013). MicroRNA biogenesis and turnover in plants. Cold Spring Harbor Symposia on Quantitative Biology 77, doi: 10.1101/sqb.2013.77.014530. 11. Yuanyuan Zhao, Beixin Mo, and Xuemei Chen. (2012). Mechanisms that impact microRNA stability in plants. RNA Biology 9, 1218-1223. 12. Yun Ju Kim and Xuemei Chen. (2011). The plant Mediator and its role in noncoding RNA production. Frontiers in Biology 6(2), 125-132. 13. So Youn Won, Rae Eden Yumul, and Xuemei Chen. Small RNAs in plants: metabolism and function. The Plant Sciences, edited by Mark Tester and Rich Jorgensen. Under review.
