Xuemei Chen - US grants

This award provides financial support for the conference entitled "Gene silencing: the biology of small RNAs and the epigenome", the 24th Symposium in Plant Biology at the University of California, Riverside, to be held January 18-20, 2007. In recent years, the importance of small RNAs to both plant and animal regulatory mechanisms governing development, physiology and responses to pathogens has been established. This conference will bring together plant scientists who have research interests in the mechanisms by which small RNAs influence gene expression. Plants possess numerous small RNAs and appear to extensively exploit small RNA pathways in various processes. The symposium comprises five major topics: (I) Small RNA biogenesis and action mechanisms; (II) Small RNA function in development; (III) Small RNA function in stress and other physiological processes; (IV) Transcriptional silencing and DNA methylation;
(V) Transcriptional silencing and histone modifications. The scientific presentations will include both oral and poster sessions.

Broader Impact: The structure of the symposium has designated slots for junior researchers, including graduate students, postdocs and early career investigators, to present their research findings. To encourage participation from young scientists and teachers from small colleges, travel assistance will be offered.

We use flower development in Arabidopsis as a model to understand how cells in multicellular organisms assume their developmental fates and form distinct patterns. Our long-term goal is to identify and analyze the genetic networks and the underlying molecular mechanisms that lead to cell fate specification in the floral primordium. For a long time, three classes of floral homeotic transcription factors known as the A, B, and C genes have been the only players in floral organ identity specification. Our studies have identified a microRNA, miR172, as a translational represser of the class A gene APETALA2, indicating that posttranscriptional regulation also plays a role in flower development. In the proposed project, we will employ molecular genetic approaches to determine how miR172 fits in the regulatory circuitry governing flower development and to probe how miR172 regulates APETALA2 mRNA at the translational level. Homeotic genes that encode transcription factors act in cell fate specification in both animals and plants. Emerging evidence of microRNAs as regulators of homeotic genes in both animals and plants adds a new layer of regulation to the known transcriptional networks governing cell fate specification. The proposed research will undoubtedly provide insights into the integration of posttranscriptional and transcriptional mechanisms in developmental processes in multicellular organisms. The proposed research will also provide insights into the mechanism of microRNA-mediated translational repression of target mRNAs. Given the conserved actions of small RNAs in gene regulation in plants and animals and the potential of using small RNAs as therapeutic agents, this research will contribute to our understanding of small RNA biology in general and impact the uses of small RNAs to treat human diseases.

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) constitute two major classes of small regulatory RNAs that play crucial roles in development, metabolism, and genome organization in plants, fungi and animals. In a previous NSF funded project, the PI's group uncovered a new step in plant miRNA biogenesis methylation of the 2' OH of the 3' terminal nucleotides. The 2'-O-methyl group protects miRNAs from a uridylation activity and an exonuclease activity. In this project, the PI's and co-PI's groups will employ biochemical approaches to probe the functions of methylation and uridylation and to use genetic approaches to identify the genes encoding the uridylation enzyme and/or the exonuclease.

Small RNAs have a profound impact on an organism's development and physiology. Understanding how small RNAs are generated, modified and degraded provides the intellectual foundation to probe various biological processes involving small RNA regulation. Although how small RNAs are processed from their precursors is well understood, little is known about how they are modified or degraded. By uncovering mechanisms in small RNA modification and degradation, this research will have profound impacts on the small RNA field and will ultimately contribute to a deeper understanding of small RNA biology and to the development of small RNA-based technologies to improve agriculture. The project will also provide research opportunities for a number of undergraduate students, including ones from underrepresented minorities.

DESCRIPTION (provided by applicant): A fundamental question in developmental biology is how pluripotent stem cells are maintained and how they differentiate into various lineages in multicellular organisms. Flower development in the model plant Arabidopsis thaliana offers a great system to address key questions in stem cell biology. One of our long-term goals is to uncover the mechanisms that govern the maintenance and termination of floral stem cells. We and others have uncovered an as yet incomplete network of three transcription factors superimposed by a microRNA (miRNA)-based posttranscriptional mechanism that governs the maintenance of floral stem cells. In the proposed research, we will expand this network by identifying core components at the heart of floral stem cell regulation. We will 1) identify target genes of the two key transcription factors, 2) incorporate the newly identified floral stem cell regulators, ARF2, ARF3 and ARF4, and the small RNA that regulates the three transcription factor genes into the existing framework of floral stem cell regulation, and 3) probe the role of the Polycomb group proteins in floral stem cell termination. miRNAs are sequence-specific regulatory molecules that impact numerous biological processes including development. The trans-acting siRNAs (ta-siRNAs) are a class of plant-specific, miRNA-like small RNAs with important developmental roles. Another long-term research goal is to dissect the general mechanisms underlying the biogenesis and mode of action of small RNAs and to study how the biogenesis or activities of specific miRNAs or ta-siRNAs are modulated in development. Whereas the major framework of miRNA biogenesis is established, how miRNAs inhibit target gene expression is highly controversial at present. The field is at its very early stages of understanding how the activities of specific small RNAs are regulated in developmental contexts. A key to dissecting the mode of action of miRNAs and understanding how small RNA activity is regulated in development is to identify proteins that mediate or modulate the activities of small RNAs. We have identified two such proteins, AGO10 and AMP1 in small RNA-mediated target gene regulation. The proposed research is aimed at uncovering the molecular functions of these two proteins. The proposed work will undoubtedly provide novel insights into stem cell regulation and miRNA function. Understanding how stem cells are maintained and terminated in plants will help derive basic principles that govern stem cell biology and contribute to the ultimate use of stem cells in regenerative medicine. Due to the highly conserved mechanisms underlying miRNA biogenesis and function between plants and animals, an advance in the mechanistic understanding of miRNA function from our work will directly impact our abilities to harness the power of small RNAs to fight pathogens and human diseases. PUBLIC HEALTH RELEVANCE: Understanding how stem cells are maintained and how they differentiate is key to harnessing the power of stem cells for regenerative medicine in the future. This research will reveal major players in the transcriptional and post-transcriptional networks that govern the temporal regulation of stem cells. This research will also advance our understanding of the mode of action of miRNAs, regulatory molecules that impact all aspects of biology and whose mis-regulation is associated with human diseases.

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.

? DESCRIPTION (provided by applicant): microRNAs (miRNAs) are sequence-specific regulators of gene expression that impact almost all biological processes in diverse eukaryotes. Defects in miRNA levels or activities are associated with numerous diseases. Both biogenesis and degradation contribute to the steady-state levels of miRNAs in vivo. The basic molecular framework underlying miRNA biogenesis has been elucidated. In contrast, although studies in ciliate, algal, plant, and animal models have implicated the existence of conserved processes that degrade miRNAs and related small RNAs, such as small interfering RNAs (siRNAs) and piwi-interacting RNAs (piRNAs), the enzymes that degrade small RNAs have yet to be identified in most organisms. As such, a basic framework of miRNA degradation awaits further studies. The goal of this project is to establish such a framework. The project capitalizes on recent advances in the area of miRNA degradation made in the PI's laboratory using the Arabidopsis model. The PI's lab identified the enzymes responsible for two conserved miRNA degradation processes in eukaryotes, 3' truncation and 3' uridylation (addition of a short, U-rich tail to miRNAs). The proposed research employs a combination of genetics, genomics, and biochemical approaches to examine the activities, interdependence and concerted actions of these enzymes with the goal of establishing a general framework of miRNA degradation. The PI's lab has also gathered preliminary evidence that implicates endogenous target mimic RNAs in miRNA turnover. The project will examine how the interplay between target mimic RNAs and the general miRNA degradation machinery results in the turnover of specific miRNAs. By elucidating principles governing miRNA degradation, the project will generate far-reaching impacts. As mounting evidence points to conserved molecular mechanisms underlying miRNA degradation in diverse eukaryotes, the proposed studies using the Arabidopsis model, from which two conserved miRNA degradation processes were first described and the enzymes responsible for these processes were first identified, will establish a general framework of miRNA degradation that is likely applicable to other eukaryotes including humans. The knowledge will enrich our understanding of various biological processes that are influenced by miRNAs and enhance our ability to control miRNA abundance to treat diseases. The molecular framework of miRNA degradation is also likely applicable to siRNAs or piRNAs, which are emerging as agents that confer epigenetic memory in plants and animals, thus further broadening the impacts of the work.

PROJECT SUMMARY microRNAs (miRNAs) are sequence-specific regulators of gene expression that impact almost all biological processes in diverse eukaryotes. Defects in miRNA-based regulation lead to developmental and physiological abnormalities in both plants and animals. Thanks to nearly two decades of research, the molecular machinery responsible for miRNA biogenesis and modes of action has been uncovered. However, since most biochemical studies on miRNAs or related small interfering RNAs were performed with cell-free extracts, how the small RNA machinery interplays with the cytoskeleton remains largely unknown. Historically, miRNAs are viewed as cell-autonomous regulatory molecules, but in recent years, mounting evidence points to the existence of miRNAs in extracellular vesicles in animals as well as the movement of miRNAs between cells in plants. Despite accumulating evidence implicating miRNAs as informational molecules in cell-cell communications, the scope of biologically significant, non-cell autonomous activities of miRNAs is largely unknown, let alone the molecular mechanisms that enable, constrain, or regulate the non-cell autonomy of miRNAs. The project interrogates the scope of miRNAs serving as informational molecules in cell-cell communications and investigates the mechanisms underlying the non-cell autonomous activities of miRNAs using the Arabidopsis model. In addition to sophisticated tool sets as available resources, advantages offered by the Arabidopsis model include the well-documented, non-cell autonomous activities of a few miRNAs in intact plants and the ease to perform forward genetic screens that do not require any a priori assumptions regarding the cellular machinery for miRNA?s non-cell autonomy. A forward genetic screen from the PI?s group revealed a previously unsuspected link between microtubules and the non-cell autonomous activities of miRNAs as well as a tantalizing connection between the translation repression activities of miRNAs and their non-cell autonomous activities. The project takes advantage of the layered cell organization in roots and employs genomics approaches at single-cell-layer resolution to study how microtubules enable the non-cell autonomous activities of miRNAs. By elucidating the scope of miRNA?s non-cell autonomy, the project has the potential to change the dogma that miRNAs largely act cell-autonomously and set the paradigm that miRNAs serve as signals in cell- cell communications. Through pioneering efforts to interrogate mechanisms underlying the non-cell autonomous activities of miRNAs, the project will provide initial knowledge in this largely unknown territory and set the foundation for future studies. By revealing a role of the cytoskeleton in small RNA biology, the impacts of the project will reach beyond plant biology.

PROJECT SUMMARY The RNA m7G cap has been recognized as a capstone in RNA metabolism in eukaryotes, thanks to decades of research that uncovered the mechanisms underlying its deposition, removal, and impacts on gene expression. However, recent discoveries showing that the m7G cap is not the only RNA cap indicate that our knowledge of RNA metabolism is far from complete. Nicotinamide adenine diphosphate (NAD+) has recently emerged as an RNA cap in bacteria, yeast, and humans, and our preliminary studies show that this cap is widespread in the model plant Arabidopsis; thus, NAD+ maybe a universal RNA cap in life. Existing evidence points to a dynamic nature of this RNA modification, as enzymes that deposit and remove the NAD+ cap have been identified in bacteria, humans, and Arabidopsis. The potentially dynamic nature of this RNA modification points to its as yet unknown regulatory functions in gene expression and biological processes. As NAD+ serves critical functions in cellular redox and energy homeostasis, it is possible that NAD+ capping/decapping in RNA is both regulated by and impacts cellular redox and metabolic homeostasis. Despite its potential importance, our knowledge of the NAD+ cap is at most rudimentary. The project seeks to understand the biology of the RNA NAD+ cap using the Arabidopsis model. Based on preliminary studies that documented the existence of NAD+-capped RNAs, implicated their translational status and revealed potential decapping enzymes, the project interrogates how the NAD+ cap is deposited and removed, how the NAD+ cap impacts gene expression, and what biological processes are regulated by RNA NAD+-capping/decapping. The sophisticated molecular and genetic resources in the Arabidopsis model not only allow for the understanding of this universal RNA modification in one domain of life, but also offer advantages of studying this RNA modification in an intact, multicellular life with relative ease. Findings on the RNA NAD+ cap from this project may have far-reaching impacts in agriculture and medicine.

Plastids are ubiquitous and essential organelles in plant cells. They come in diverse types, with chloroplasts being the most well-known plastid type. Through photosynthesis, chloroplasts harvest solar energy and convert carbon dioxide into plant biomass, and thus sustain almost all life on Earth. In fact, the functions of plastids are not limited to photosynthesis ? other types of plastids exist and perform a variety of essential functions in development, metabolism, signaling, and immunity in plants. Plastids and their diverse functions can be harnessed for the benefit of humans and the environment. However, our knowledge of plastids is severely limited by current technologies. Each plant cell harbors tens to hundreds of plastids and current approaches can either study a few plastids at a time, such as by microscopy, or millions of them in bulk, such as by molecular techniques. The former lacks efficiency while the latter averages potentially distinct plastid types. The proposed experiments are aimed at establishing a new technology to study plastids, namely single-plastid RNA sequencing (spRNA-seq). This technology determines the molecular signatures of hundreds of thousands of individual plastids in one experiment, making it possible to efficiently and fully understand plastid diversity and how they function. The technology is expected to revolutionize research on plastids and will generate novel insights into how plastids affect plant biology, ecology, and evolution. This knowledge can be used to harness the power of plastids for the betterment of life on Earth.

The PIs of this collaborative project plan to use plastids from Arabidopsis thaliana and Pisum sativum, as both technical controls as well as biologically comparative models, to establish the spRNA-seq method. The project comprises three integrative components. First, a genomics strategy is designed, optimized, and implemented to achieve spRNA-seq that is reproducible, cost-effective, and user-friendly. In particular, plastids will be isolated and a split-and-pool strategy will be employed to enable in-plastid combinatorial barcoding during library preparation, with each plastid being identified bioinformatically through the unique combination of barcodes. Next, a suite of computational and statistical approaches will be applied to evaluate the technical outcomes of spRNA-seq, and more importantly, to glean insights into new plastid types through transcriptomic heterogeneity. As the plastid transcriptome is heavily molded by posttranscriptional mechanisms such as splicing, editing, and endonucleolytic and exonucleolytic processing, potential inter-plastid heterogeneity with respect to these posttranscriptional events will be interrogated. Finally, single-molecule RNA in situ hybridization will be deployed for validation of plastid heterogeneity. This complementary approach adds spatial resolution to provide further insights into plastid heterogeneity within a cell or tissue. Further, the project will train the next generation of scientists, including underrepresented minority students at University of California Riverside, in critical thinking, experimental design and execution, and scientific communications. The research team will disseminate this empowering technology through publications, conference presentations, and training videos to propel scientific discovery.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.