SMALL RNAs IN RICE AND MAIZE

1. SMALL RNAs IN RICE AND MAIZE Lewis Bowman1, Vicki Vance1, Rebecca Bullard-Dillard2, Cameron Johnson3, *, Alex Adai3, *, Venkatesan Sundaresan3, * 1 University of South Carolina, Columbia, SC, 2Claflin University, Orangeburg, SC, 3University of California, Davis, CA, * Bioinformatics Expertise PROJECT SUMMARY Aim 1C.Develop a project website. A web interface to all the data is being established to include all small RNA sequences, the number and location of miRNA genes, structure of miRNA precursors, predicted miRNA targets and the pattern of miRNA and other small RNA accumulation during development and in response to stress. Scientific Objectives. Project Objectives: Endogenous small regulatory RNAs, including short interfering RNAs (siRNAs), microRNAs (miRNAs) and other related small RNAs, mediate development and responses to stress in plants as in other eukaryotic organisms. Knowledge of the number, sequence and expression patterns of such small RNAs as well as their regulatory targets is essential in order to understand the basic biology of plants as well as to develop high yielding, stress resistant crops. The major goal is to extensively analyze the sequences and expression patterns of all or nearly all of the small RNAs expressed in two important crop plants, rice and maize. The first aim of this project is to establish a small RNA database and identify miRNAs and other functional small RNAs in both rice and maize. Targets of small RNAs will also be identified and validated. The second aim is to measure the global expression pattern of miRNAs and other small RNAs during development and as a function of various stresses. The third aim is to map the rice and maize miRNA transcription units. A web interface to all the data will be established to include all small RNA sequences, the number and location of miRNA genes, structure of miRNA precursors, predicted miRNA targets and the accumulation pattern of miRNAs and other small RNAs during development and in response to stress. Experimental Approaches: High-throughput pyrosequencing from 454 Biosciences is being used to establish a database of small RNAs from rice and maize as well as profile small RNAs from selected developmental stages and environmental stress conditions. We have sequenced 390,002 maize and 294,802 rice small RNAs from various tissues and developmental stages/stress conditions and have established a cereal small RNA database, including both rice and maize sequences. The web interface to the data is in the final debugging stages and should be released to the community within the next few months. Various criteria are being employed to identify new miRNAs among the sequenced small RNAs. Predicted targets of new miRNAs are being validated using RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The patterns of miRNA and small RNA accumulation will be determined by direct 454 sequence profiling. Initial studies seek to identify the pattern of small RNA accumulation in vegetative and reproductive meristems. Forthcoming analysis of the initial 454 survey of a variety of stress conditions will guide our choice of particular stress conditions. A total of 21 additional pyrosequencing runs will be performed to profile small RNAs. In addition, the considerable depth of sequencing will facilitate the identification of the functions of various classes of small RNAs. The structure of selected miRNA precursors in both rice and maize will be elucidated using RACE techniques. Information/Materials to be Generated: A project website will provide access to: 1) an extensive database of small RNA sequences and miRNA precursors accessible via a genome browser, 2) a candidate miRNA-target dataset and 3) the pattern of expression of small RNAs in various developmental stages and stress conditions. Finally, due to the connection between silenced chromatin and 24 nt small RNAs, a major end result of the analysis will be a preliminary epigenetic map of the rice and maize genomes (the “epigenome”). Figure 1. Genome browser. Mapped smRNAs are grouped in individual size classes. Tracks are also included for Rfam miRNAs and precursors, smRNAs capable of forming miRNA/miRNA* duplexes as in miRNA precursors (biased fold duplexes), and TIGR cDNAs PROGRESS ON SPECIFIC AIMS Figure 2A. Potential small RNA targets. A link from each smRNA leads to predicted mRNA targets. Initial smRNA-target duplexes are predicted using FASTH which returns results based on thermal stabilities. These FASTH output is then processed to enable ranking of the duplexes based on known characteristics of validated miRNA-target duplexes. The database can also be queried independently using transcript IDs. Figure 2B. Conserved miRNA targets sites. The transcripts checked Figure 2A are aligned to facilitate identification of conserved target sites. Specific Aim 1A.. Generate a small RNA database from Rice and Maize. Small RNAs were isolated from a variety of tissues at different developmental stages and fromplants stressed by various pathogens and different types of abiotic insults. Figure 2C. smRNAs targeting a single transcript. Following a link (FASTH-mRNAs) on the predicted targets page (figure 2A) returns a list of smRNAs targeting that mRNA. The greater color intensity in the smRNA boxes is an indication of a better alignment score. Specific Aim 1B. Identification of miRNAs in the small RNA sequence populations. Several criteria are currently being used to identify new miRNAs. These include sequence conservation, capacity to form fold back precursor, presence of miRNA*, relatively low repetition in the genome, the presence of a 5`U residue and absence or near absence of small RNA derived from the opposite strand. The dependence of potential new rice miRNAs on DCL1 or DCL4 will also be determined using knockdowns of these genes. Predicted targets of new miRNAs are being validated using RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). Identification of other types of functional RNAs will also be undertaken. Specific Aim 2. The patterns of miRNA and small RNA accumulation during development and as a function of stress will be determined by direct 454 sequence profiling. Initial studies are focusing on the pattern of small RNA accumulation in vegetative and reproductive meristems in rice and maize, and library construction for many of the tissues is underway.