Small RNAs and Arabidopsis. A Fast Forward Look

First, let's take a quick look back to a few formative events leading to the blur of activity that now defines the small RNA field. In 1993, Victor Ambros and colleagues (Lee et al., 1993) identified the founding member of a large class of genetic regulators (later termed microRNAs [miRNAs], approximately 22 nucleotides) with the pioneering discovery of the small RNA product of the Caenorhabditis elegans heterochronic gene lin-4. It was not until many years later that the broad relevance of small RNAs was realized. In 1999, David Baulcombe's group (Hamilton and Baulcombe, 1999) made the seminal discovery that short interfering RNAs (siRNAs; approximately 21–24 nucleotides) were associated with posttranscriptional gene silencing triggered by transgenes and viruses in plants, laying the conceptual foundation for groundbreaking discoveries about the RNA interference (RNAi) biochemical pathway. The discoveries of miRNA and siRNA biogenesis and effector pathways, the identification of hundreds of miRNA genes (plants and animals) and diverse endogenous siRNA-generating loci (plants and fungi), and the finding of links between RNAi and heterochromatin formation (plants and fungi) transformed our view of RNA-based regulation in eukaryotes (Bartel, 2004). This has been an exciting time in the RNA regulatory field, in part due to the remarkable convergence of developmental biologists, RNA and chromatin biochemists, geneticists, and many others working in animal, plant, fungal, and protist systems. From a plant biologist's perspective, I derive great satisfaction with the frontline contributions plant experimental systems provide to this field. Further fueling both excitement and satisfaction is the seemingly unlimited application of RNAi and small RNAs to functional genomics, medicine, and agriculture.

I feel at risk, therefore, projecting a future outlook for this fast-moving field. Statements concerning the most important questions to address and the technology needed to answer these questions may be entirely obsolete by the time this article appears in print. Nevertheless, I offer the following four areas as fertile ground for discovery in plant small RNA biology.

INTEGRATION OF SMALL RNA PATHWAYS IN DEVELOPMENT AND ADAPTIVE RESPONSES

A large proportion of plant miRNA families, and some trans-acting (ta) siRNAs, function as negative regulators of mRNAs coding for transcription factors with roles in development (Jones-Rhoades and Bartel, 2004). For example, miR160 and miR167 each target clades within the auxin response factor family of transcription factors. Several others miRNA target genes encode factors controlling meristem function and patterning. Disruption of negative regulation by miRNAs, through mutation of target sites or miRNA sequences, frequently results in developmental phenotypes (Baulcombe, 2004). However, the mechanisms whereby miRNAs and ta-siRNAs are integrated into developmental or signaling pathways are understood poorly. To what extent do these small RNAs trigger decisive or committed steps in cell differentiation? Do they also function as constitutive negative regulators to direct or reinforce the “off” state of target genes? How are miRNA genes controlled?

It is clear that dynamic, cell-specific imaging of expression or activity patterns of specific miRNAs and ta-siRNAs, target mRNAs, and downstream genes over time is needed to place these small RNAs into spatial and temporal contexts during development. Two major types of resources are either under development or in early deployment to address these problems. The first is sensor technology, in which fluorescent proteins serve to reveal cell-specific patterns of miRNA gene expression and targeting activity. For example, sensor constructs containing miRNA target sequences are particularly useful as reporters of miRNA activity in situ (Parizotto et al., 2004). The second is microarray technology (or comparable methods) to analyze miRNAs, targets, and downstream genes in parallel. Virtual in situ expression analysis, as described by the Benfey laboratory (Birnbaum et al., 2003), would appear particularly well suited to understand integration of small RNAs in developmental pathways. Additionally, genetic resources in which entire miRNA gene families (rather than single, potentially redundant family members) are inactivated are needed.

EVOLUTION AND SPECIALIZATION OF SMALL RNA PATHWAYS

An early eukaryote likely possessed genes encoding a repertoire of RNAi factors, including Dicer-like (DCL), Argonaute, and RNA-dependent RNA polymerase proteins. The DCL protein contained two RNaseIII-like domains for double-stranded RNA processing, whereas the Argonaute protein likely possessed an RNaseH-like domain. Families for each of these three factors expanded in plants after divergence from other eukaryotic lineages, providing genes for specialization of miRNA, ta-siRNA, heterochromatin-associated RNAi, and antiviral RNAi pathways (Baulcombe, 2004). However, functions still remain to be assigned to the majority of the 10 Argonaute members in Arabidopsis (Arabidopsis thaliana) and some of the DCL factors. The Argonautes are particularly interesting. Functions in miRNA and siRNA-directed target cleavage have been assigned for only two family members (AGO1 and AGO4), while a third (ZIPPY) was functionally assigned as a factor in juvenile-to-adult phase transitions (Baulcombe, 2004). Why are there so many more AGO factors than known miRNA- and siRNA-dependent biochemical pathways? My guess is that there is far more pathway specialization than we currently realize.

In addition to small RNA biogenesis and effector factor families, the small RNA-generating loci themselves belong to gene families and are still evolving. Sequence duplication events resulting in foldback or hairpin transcripts appear to be one mechanism whereby new small RNA regulators with novel specificity can arise (Allen et al., 2004). Are there other mechanisms? Analysis of small RNAs and small RNA-generating loci in close relatives of Arabidopsis will be informative about the frequency of formation of potential new miRNA genes. Additionally, analysis of siRNAs in synthetic polyploids resulting from combinations of genomes may shed light on mechanisms of large-scale heterochromatin formation through RNAi-dependent processes.

INTEGRATION OF SMALL RNA COMPONENTS, EFFECTOR COMPLEXES, AND CELLULAR PATHWAYS

Some of the best work to define biochemical components and activities associated with small RNA formation and effector pathways comes from analysis of fly extracts and other animal-based in vitro systems (Bartel, 2004). Relatively little plant RNAi biochemistry has progressed to the point of publication. While there are clearly some interesting biochemical issues that need to be addressed in plants, such as how different DCLs catalyze formation of different classes of small RNAs, there are many technical barriers that limit progress. Analysis of complexes from wild-type and mutant Arabidopsis plants or cells through immunoprecipitation or other affinity tags may offer a path around some of these barriers.

Another interesting set of questions concerns how RNAi complexes integrate with other cellular processes. For example, while it is clear that a set of specialized RNAi components (DCL3, RDR2, AGO4) participate in heterochromatin formation at many loci (Zilberman et al., 2003; Xie et al., 2004), exactly how these factors integrate with DNA and histone modification pathways is not clear. What is the role of RNA polymerase IV (SDE4) in heterochromatin-associated RNAi (Herr et al., 2005; Onodera et al., 2005)? Conceptually similar types of questions remain for each miRNA- and siRNA-related pathway and the cellular processes that they affect.

SYMPLASTIC DYNAMICS OF SMALL RNA PATHWAYS

Among the least understood areas of RNAi biology in plants concerns the intercellular mobility of small RNAs and small RNA-containing complexes. We have known for nearly a decade that sequence-specific information during RNA silencing is transported over short (cell-to-cell) and long (phloem-dependent) distances (Baulcombe, 2004). The antiviral response appears to depend, in part, on systemic transport of RNAi signals. However, the mechanistic basis of this pathway as well as the relevance of transport of active miRNAs and siRNAs during growth and development remain ill defined. Genetic data supporting identification and function of components of these transport pathways are absent. Clearly, creative genetic screens for informative mutants with specific defects in the RNA transport system(s) are desperately needed.

In addition to these fundamental areas, the plant-based biotechnology industry is poised to deliver novel products that involve small RNAs. The predictability and broad utility of directed silencing using available RNAi technology and the potential of other technologies that deploy different small RNA pathways suggest that RNAi-conditioned phenotypes may proliferate in agricultural products. The limitations for industry appear more related to social forces rather than scientific potential.