Large-scale sequencing of plant small RNAs.

Deep sequencing technologies have become very powerful tools in the identification and quantification of small RNAs involved in gene regulation. Small interfering RNA (siRNA) and miRNA are two classes of DCL-dependent small RNAs known to affect phenotype, developmental regulation, and various traits in plants. These small RNAs function by selectively repressing gene expression mainly by guiding cleavage, resulting in degradation of target transcripts. In this chapter, we describe a method for preparation of 5(')-phosphate-dependent small RNA libraries, a hallmark of RNase III-like DCL products, for high-throughput sequencing, and recommendations for small RNA analysis. This method is useful for determining small RNA involvement in critical pathways in plants, identifying and quantifying novel small RNAs, and examining small RNA global expression patterns.

miRNAs in the biogenesis of trans-acting siRNAs in higher plants.

Multicellular eukaryotes utilize many complex small RNA mechanisms to regulate gene expression from DNA modifications to RNA stability. RNA interference also regulates exogenous gene expression by degrading invading pathogen RNAs or preventing expression of foreign DNA incorporated into the host genome. Here we review the mechanisms for trans-acting (ta)-siRNA biogenesis and function, including pathways that utilize components of the miRNA and transitive RNAi defense. There are several distinguishing features of ta-siRNA pathways including the requirement for a miRNA-guided cleavage event that sets a processing register, RDR6 dependent dsRNA production, and DCL4 dependent processing to create unique, phased 21 nucleotide small RNAs. These phased small RNAs function to suppress target genes that only show similarity at the ta-siRNA recognition site, and act in trans to repress expression non-cell autonomously of specific target genes. Since the advent of high throughput sequencing technologies, phased siRNAs have been identified in a number of organisms [Heisel SE, Zhang Y, Allen E, Guo L, Reynolds TL, Yang X, et al. Characterization of unique small RNA populations from rice grain. PLoS One 2008;3:e2871. Zhao T, Li G, Mi S, Li S, Hannon GJ, Wang XJ, et al. A complex system of small RNAs in the unicellular green alga Chlamydomonas reinhardtii. Genes Dev 2007;21:1190-203. Johnson C, et al. Clusters and superclusters of phased small RNAs in the developing inflorescence of rice. Genome Res 2009;19:1429-40. Zhu QH, Spriggs A, Matthew L, Fan L, Kennedy G, Gubler F, et al. A diverse set of microRNAs and microRNA-like small RNAs in developing rice grains. Genome Res 2008;18:1456-65. Howell MD, Fahlgren N, Chapman EJ, Cumbie JS, Sullivan CM, Givan SA, et al. Genome-wide analysis of the RNA-DEPENDENT RNA POLYMERASE6/DICER-LIKE4 pathway in Arabidopsis reveals dependency on miRNA- and ta-siRNA-directed targeting. Plant Cell 2007;19:926-42.]. These include transcripts generated either from non-protein-coding or protein-coding transcripts, long imperfect dsRNA or through an unknown mechanism; therefore some of these may not necessarily be classified as canonical ta-siRNAs.

Pattern formation via small RNA mobility.

MicroRNAs and trans-acting siRNAs (ta-siRNAs) have important regulatory roles in development. Unlike other developmentally important regulatory molecules, small RNAs are not known to act as mobile signals during development. Here, we show that low-abundant, conserved ta-siRNAs, termed tasiR-ARFs, move intercellularly from their defined source of biogenesis on the upper (adaxial) side of leaves to the lower (abaxial) side to create a gradient of small RNAs that patterns the abaxial determinant AUXIN RESPONSE FACTOR3. Our observations have important ramifications for the function of small RNAs and suggest they can serve as mobile, instructive signals during development.

AGO1-miR173 complex initiates phased siRNA formation in plants.

MicroRNA (miRNA)-guided cleavage initiates entry of primary transcripts into the transacting siRNA (tasiRNA) biogenesis pathway involving RNA-DEPENDENT RNA POLYMERASE6, DICER-LIKE4, and SUPPRESSOR OF GENE SILENCING3. Arabidopsis thaliana TAS1 and TAS2 families yield tasiRNA that form through miR173-guided initiation-cleavage of primary transcripts and target several transcripts encoding pentatricopeptide repeat proteins and proteins of unknown function. Here, the TAS1c locus was modified to produce synthetic (syn) tasiRNA to target an endogenous transcript encoding PHYTOENE DESATURASE and used to analyze the role of miR173 in routing of transcripts through the tasiRNA pathway. miR173 was unique from other miRNAs in its ability to initiate TAS1c-based syn-tasiRNA formation. A single miR173 target site was sufficient to route non-TAS transcripts into the pathway to yield phased siRNA. We also show that miR173 functions in association with ARGONAUTE 1 (AGO1) during TAS1 and TAS2 tasiRNA formation, and we provide data indicating that the miR173-AGO1 complex possesses unique functionality that many other miRNA-AGO1 complexes lack.

Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation.

Trans-acting siRNA form through a refined RNAi mechanism in plants. miRNA-guided cleavage triggers entry of precursor transcripts into an RNA-DEPENDENT RNA POLYMERASE6 pathway, and sets the register for phased tasiRNA formation by DICER-LIKE4. Here, we show that miR390-ARGONAUTE7 complexes function in distinct cleavage or noncleavage modes at two target sites in TAS3a transcripts. The AGO7 cleavage, but not the noncleavage, function could be provided by AGO1, the dominant miRNA-associated AGO, but only when AGO1 was guided to a modified target site through an alternate miRNA. AGO7 was highly selective for interaction with miR390, and miR390 in turn was excluded from association with AGO1 due entirely to an incompatible 5' adenosine. Analysis of AGO1, AGO2, and AGO7 revealed a potent 5' nucleotide discrimination function for some, although not all, ARGONAUTEs. miR390 and AGO7, therefore, evolved as a highly specific miRNA guide/effector protein pair to function at two distinct tasiRNA biogenesis steps.

Genome-wide analysis of the RNA-DEPENDENT RNA POLYMERASE6/DICER-LIKE4 pathway in Arabidopsis reveals dependency on miRNA- and tasiRNA-directed targeting.

Posttranscriptional RNA silencing of many endogenous transcripts, viruses, and transgenes involves the RNA-DEPENDENT RNA POLYMERASE6/DICER-LIKE4 (RDR6/DCL4)-dependent short interfering RNA (siRNA) biogenesis pathway. Arabidopsis thaliana contains several families of trans-acting siRNAs (tasiRNAs) that form in 21-nucleotide phased arrays through the RDR6/DCL4-dependent pathway and that negatively regulate target transcripts. Using deep sequencing technology and computational approaches, the phasing patterns of known tasiRNAs and tasiRNA-like loci from across the Arabidopsis genome were analyzed in wild-type plants and silencing-defective mutants. Several gene transcripts were found to be routed through the RDR6/DCL4-dependent pathway after initial targeting by one or multiple miRNAs or tasiRNAs, the most conspicuous example of which was an expanding clade of genes encoding pentatricopeptide repeat (PPR) proteins. Interestingly, phylogenetic analysis using Populus trichocarpa revealed evidence for small RNA-mediated regulatory mechanisms within a similarly expanded group of PPR genes. We suggest that posttranscriptional silencing mechanisms operate on an evolutionary scale to buffer the effects of rapidly expanding gene families.

High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes.

In plants, microRNAs (miRNAs) comprise one of two classes of small RNAs that function primarily as negative regulators at the posttranscriptional level. Several MIRNA genes in the plant kingdom are ancient, with conservation extending between angiosperms and the mosses, whereas many others are more recently evolved. Here, we use deep sequencing and computational methods to identify, profile and analyze non-conserved MIRNA genes in Arabidopsis thaliana. 48 non-conserved MIRNA families, nearly all of which were represented by single genes, were identified. Sequence similarity analyses of miRNA precursor foldback arms revealed evidence for recent evolutionary origin of 16 MIRNA loci through inverted duplication events from protein-coding gene sequences. Interestingly, these recently evolved MIRNA genes have taken distinct paths. Whereas some non-conserved miRNAs interact with and regulate target transcripts from gene families that donated parental sequences, others have drifted to the point of non-interaction with parental gene family transcripts. Some young MIRNA loci clearly originated from one gene family but form miRNAs that target transcripts in another family. We suggest that MIRNA genes are undergoing relatively frequent birth and death, with only a subset being stabilized by integration into regulatory networks.

Regulation of AUXIN RESPONSE FACTOR3 by TAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis.

MicroRNAs (miRNAs) and trans-acting siRNAs (ta-siRNAs) in plants form through distinct pathways, although they function as negative regulators of mRNA targets by similar mechanisms . Three ta-siRNA gene families (TAS1, TAS2, and TAS3) are known in Arabidopsis thaliana. Biogenesis of TAS3 ta-siRNAs, which target mRNAs encoding several AUXIN RESPONSE FACTORs (including ARF3/ETTIN and ARF4 ) involves miR390-guided processing of primary transcripts, conversion of a precursor to dsRNA through RNA-DEPENDENT RNA POLYMERASE6 (RDR6) activity, and sequential DICER-LIKE4 (DCL4)-mediated cleavage events. We show that the juvenile-to-adult phase transition is normally suppressed by TAS3 ta-siRNAs, in an ARGONAUTE7-dependent manner, through negative regulation of ARF3 mRNA. Expression of a nontargeted ARF3 mutant (ARF3mut) in a wild-type background reproduced the phase-change phenotypes detected in rdr6-15 and dcl4-2 mutants, which lose all ta-siRNAs. Expression of either ARF3 or ARF3mut in rdr6-15 plants, in which both endogenous and transgenic copies of ARF3 were derepressed, resulted in further acceleration of phase change and severe morphological and patterning defects of leaves and floral organs. In light of the functions of ARF3 and ARF4 in organ asymmetry, these data reveal multiple roles for TAS3 ta-siRNA-mediated regulation of ARF genes in developmental timing and patterning.