Small RNAs hit the big time

Small RNAs have exploded on the scene as ubiquitous, versatile repressors of gene expression in plants, animals and many fungi. These small RNAs (21–26 nt) are cleaved from a double-stranded (ds) RNA and induce effects through homologous sequence interactions. Small RNAs come in various forms: microRNAs (miRNAs), heterochromatizing RNAs, tiny noncoding RNAs, short interfering (si) RNAs, natural antisense short interfering RNAs (nat-siRNAs) and small temporal (st) RNAs. They control mRNA stability or translation, or target epigenetic modifications of DNA or histones at specific regions of the genome. Small RNAs have established a new paradigm for understanding eukaryotic gene regulation. The pivotal discovery of dsRNA and its role in RNA silencing in the cell was acknowledged by the award of the Nobel Prize in Medicine or Physiology in 2006 to Andrew Fire (Standford University, CA, USA) and Craig Mello (University of Massachusetts, Worcester, MA, USA), 8 yr after the seminal publication (Fire et al., 1998). In January 2007, the University of California at Riverside hosted a meeting to discuss the recent advances in small RNAs and epigenetics within the plant science field. In this article, we present the highlights of that meeting, summarize the current status of the field in plants, and point out areas yet to be explored. ‘… Arabidopsis currently has the largest data set of small RNAs of any species with 300 000 unique small RNAs …’ Appreciation of the number and complexity of small RNAs in plants and other organisms has been accelerated by revolutionary advances in high-throughput nanosequencing technology (Fig. 1). Pyrosequencing and sequencing by synthesis methods now allow 500 000 small RNA sequences to be generated in a single experiment (Margulies et al., 2005). This is typified by Arabidopsis, which currently has the largest data set of small RNAs of any species, at 300 000 unique small RNAs and growing (Henderson et al., 2006; Lu et al., 2006; Rajagopalan et al., 2006; Fahlgren et al., 2007). These sequences, along with current small RNA sequencing projects in model organisms and agronomically important species such as Physcomitrella patens, Chlamydomonas reinhardtii, rice (Oryza sativa) and maize (Zea mays), will provide a valuable resource for the scientific community to exploit (Axtell et al., 2006; Johnson et al., 2006; Qi Yijun, National Institute of Biological Sciences, Beijing, China; David Baulcombe, The Sainsbury Laboratory (TSL), John Innes Centre (JIC), Norwich, UK; Pam Green, Delaware Biotechnology Institute (DBI), Newark, DE, USA). An ambitious project underway at the Blake Meyers (DBI, USA) and Pam Green laboratories is to sequence small RNAs from about 30 plant species, a task that will undoubtedly provide a rich resource for the community. The number of sequenced unique small RNAs from Arabidopsis thaliana. The asterisk indicates the first small RNA data set published using picolitre pyrosequencing technology. Beyond cataloguing the small RNA populations, researchers are using small RNA sequence data to ask questions about the evolution and biogenesis of small RNAs. Jim Carrington (Oregon State University, Corvallis, OR, USA) presented data to support the hypothesis that several nonconserved miRNAs in Arabidopsis are the evolutionary intermediates between genome rearrangements and a miRNA (Fahlgren et al., 2007). This work highlights the power of bioinformatic analysis of large data sets. The sequencing of small RNAs (sRNAs) from Arabidopsis has led to the identification of abundant 21- and 24-nt RNAs. Twenty-one-nucleotide RNAs target cleavage of mRNAs and guide the cleavage and subsequent phased production of transacting siRNAs (tasiRNAs). The production of 21-nt small RNAs has also been linked to cell-to-cell signalling, although longer dsRNA movement cannot be ruled out (Dunoyer et al., 2005). Twenty-four-nucleotide RNAs are required for RNA-directed DNA methylation and heterochromatinization in Arabidopsis (Qi et al., 2006); however, the exact mechanism of this is still to be elucidated. Large-scale sequencing of heterochromatic small RNAs and the genome-wide description of DNA methylation in Arabidopsis suggest where the 24-nt RNAs may function; however, whether the sRNAs are a cause or a consequence of DNA methylation is still to be determined (Zhang et al., 2006; Steve Jacobsen, University of California, Los Angeles, CA, USA). An open question is whether 21-nt, 24-nt or longer RNAs are involved in long-distance spreading of silencing. Elegant grafting experiments in a transgenic Arabidopsis system hint at longer dsRNA as a phloem-transmitted signal (Bernard Carroll, University of Queensland, Brisbane, Australia). In this model, the transported dsRNA is then incorporated in cells that receive the signal into a 24-nt small RNA generating pathway for RNA silencing. Small RNAs (21–24 nt) are presumed to be generated through Dicer cleavage of a long dsRNA intermediate. Recent work has teased apart the overlapping functions of the four Arabidopsis Dicer-like proteins (Herve Vaucheret, INRA, Versailles, France; Olivier Voinnet, Institut de Biologie Moléculaire des Plantes, Strasbourg, France). Intriguingly, recent published and unpublished data sets have also identified larger (26, 28 and > 30 nt) small RNAs (Katiyar-Agarwal et al., 2006; Hailing Jin, University of California, Riverside, CA, USA). How are these larger small RNAs generated? One hypothesis is that they are not generated through Dicer cleavage, but rather through the interrupted action of an RNA-dependent RNA polymerase, as has been shown in Caenorhabditis elegans (Pak & Fire, 2006; Sijen et al., 2006). The incredible growth of small RNA data presents several challenges for the near future. Technological advances have allowed massive data sets to be generated, but there is an immediate requirement for a central database for depositing small RNA sequences. There is also a need for generation and sharing of bioinformatic tools for analysis. Bioinformatics will be integral to readily manipulating large data sets, allowing cross-species comparisons, and assaying genome-wide changes in small RNAs. Another challenge of the field is a detailed description of small RNAs at the organ and ultimately cellular levels, as well as elucidation of how small RNAs respond to environmental changes. A step towards these goals is the deep sequencing of small RNAs from heat- and salt-stressed Arabidopsis by the Meyers and Green laboratories. Another interesting area is natural variation in methylation patterns. Eric Richards (Washington State University, St Louis, WA, USA) has taken the approach to look for natural variation in methylation patterns in Arabidopsis and to uncover genetic elements involved in this process. Arabidopsis contains four Dicer-like proteins, six RNA-dependent RNA polymerases, and 10 Argonaute proteins. This diversity allows for complexity in small RNA biogenesis, and emerging data suggest that these proteins can act as modules to be interchanged to generate functional diversity. An example of modularity can be found in the newly identified miRNAs that are Dicer-like (DCL)4 dependent rather than DCL1 dependent (Rajagopalan et al., 2006) or in a transgene silencing system in which dsRNA production by Polymerase (Pol)IV is uncoupled from downstream processing by DCL3 and Argonaute (AGO)4, as with endogenous siRNAs, and instead is linked to DCL4 and AGO2 (David Baulcombe, TSL, JIC, Norwich, UK). These findings challenge our current view of discrete, linear small RNA pathways. Histone modifications have also been linked to small RNAs in plants (Onodera et al., 2005; Huettel et al., 2006). The quest for a full description of histone modifications – how they are added and removed, and exactly how they impact on gene regulation – is an emerging area. Steve Jacobsen (University of California, Los Angeles, CA, USA) has followed on from his genomic DNA methylation work to describe genome-wide histone methylation in Arabidopsis. He has shown that H3K27 trimethylation is generally present in small domains and is anticorrelated with small RNA accumulation. More detailed analysis of small RNA-directed histone modification was presented by researchers of silencing in Schizosaccharomyces pombe (Danesh Moazed, Harvard University, Boston, MA, USA; Robert Martienssen, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA). Perhaps the most important challenge facing the small RNA community is linking cellular small RNAs to biological function. Small RNAs have been implicated in viral defence for several years. Recent work has expanded the role of sRNAs into defence against bacterial pathogens, in particular how pathogen miRNAs and host miRNAs contribute to pathogenesis and defence (Katiyar-Agarwal et al., 2006; Navarro et al., 2006). Small RNAs also play a role in response to abiotic stresses. Overlapping, convergent transcripts have the ability to form dsRNA and to be processed into siRNAs, leading to regulation of one of the transcripts (Borsani et al., 2005). This has been demonstrated to be important for salt tolerance in Arabidopsis. Small RNA pathways may also be implicated in control of flowering (Caroline Dean, JIC, Norwich, UK). Further analysis of the abundant small RNA sequence data will undoubtedly lead to an increased understanding of the complex interactions of small RNA-generating loci. We are only starting to appreciate the role of small RNAs in the cell and many questions still exist. How dynamic are these small RNA populations? What are the diurnal changes, developmental changes, environmentally induced changes and microbe-induced changes of small RNAs? How did the different types of small RNAs evolve? It is an exciting time for small RNA biologists; this was demonstrated by the inspiring work presented at the 24th Symposium in Plant Biology at University of California Riverside. We apologize to those meeting contributors whose work we did not mention because of space limitations. IS was awarded a Marie Curie International Incoming Fellowship and an Epigenome NoE travel award. RM was awarded a Marshall Fellowship and CM was supported by a Commonwealth Scholarship and by the Natural Sciences and Engineering Research Council of Canada. The Sainsbury Laboratory is funded by the Gatsby Charitable Trust.

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