Plants and animals use small RNAs (microRNAs [miRNAs] and siRNAs) as guides for posttranscriptional and epigenetic regulation. In plants, miRNAs and trans-acting (ta) siRNAs form through distinct biogenesis pathways, although they both interact with target transcripts and guide cleavage. An integrated approach to identify targets of Arabidopsis thaliana miRNAs and ta-siRNAs revealed several new classes of small RNA-regulated genes, including conventional genes such as Argonaute2 and an E2-ubiquitin conjugating enzyme. Surprisingly, five ta-siRNA-generating transcripts were identified as targets of miR173 or miR390. Rather than functioning as negative regulators, miR173- and miR390-guided cleavage was shown to set the 21-nucleotide phase for ta-siRNA precursor processing. These data support a model in which miRNA-guided formation of a 5' or 3' terminus within pre-ta-siRNA transcripts, followed by RDR6-dependent formation of dsRNA and Dicer-like processing, yields phased ta-siRNAs that negatively regulate other genes.

Plants with altered microRNA metabolism have pleiotropic developmental defects, but direct evidence for microRNAs regulating specific aspects of plant morphogenesis has been lacking. In a genetic screen, we identified the JAW locus, which produces a microRNA that can guide messenger RNA cleavage of several TCP genes controlling leaf development. MicroRNA-guided cleavage of TCP4 mRNA is necessary to prevent aberrant activity of the TCP4 gene expressed from its native promoter. In addition, overexpression of wild-type and microRNA-resistant TCP variants demonstrates that mRNA cleavage is largely sufficient to restrict TCP function to its normal domain of activity. TCP genes with microRNA target sequences are found in a wide range of species, indicating that microRNA-mediated control of leaf morphogenesis is conserved in plants with very different leaf forms.Although much is known about how organs acquire their particular fate, we are only starting to learn how organs are sculpted, even if they are just flat sheets such as wings or leaves. An elegant study recently demonstrated that making a flat organ is not a trivial problem: snapdragon leaves are normally flat, but they become crinkly in plants lacking the CINCINNATA (CIN) gene 1 . In cin mutants, differential regulation of cell division across the leaf is disturbed, causing negative leaf curvature. CIN RNA itself is expressed in a dynamic pattern, in front of and perhaps overlapping the mitotic arrest zone, suggesting a direct role of CIN in regulating leaf morphogenesis.Although it is unknown how expression of CIN, which encodes a TCP transcription factor 2 , is regulated, a specific RNA pattern can result from differential transcription or changes in transcript stability. A post-transcriptional mechanism that has only recently been recognized is that of plant mRNA cleavage initiated by partially or fully complementary microRNAs (miRNAs) 3,4 . The mechanism of cleavage is similar, or identical, to cleavage guided by short interfering RNAs (siRNAs) 5 .The double-stranded ribonucleases Dicer in animals and DICER-LIKE1 (DCL1) in plants process miRNAs-which are usually 21-22 nucleotides long-from longer precursor RNAs with fold-back structure 4,6,7 . Additional factors required for accumulation of miRNAs include members of the Argonaute family and HEN1 protein 8,9 . The importance of miRNAs for plant development is supported by the abnormalities seen in several mutants or transgenic plants with general defects in miRNA accumulation or activity [9][10][11][12] . However, although biochemical studies have demon- Fig. 2b). b, Seedlings, individual leaves and leaf rosettes of Columbia wild-type and jaw-1D plants. Leaves were mounted between glass plates and illuminated from below. Dark green areas indicate overlapping leaf parts after flattening. c, Expression changes of TCP genes in jaw-1D estimated from Affymetrix arrays (grey bars) or from RT-qPCR (black bars). See Supplementary Information for absolute values. NP, termed 'not present' by MAS software. Note th...

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.

The molecular basis for virus-induced disease in plants has been a long-standing mystery. Infection of Arabidopsis by Turnip mosaic virus (TuMV) induces a number of developmental defects in vegetative and reproductive organs. We found that these defects, many of which resemble those in miRNA-deficient dicer-like1 (dcl1) mutants, were due to the TuMV-encoded RNA-silencing suppressor, P1/HC-Pro. Suppression of RNA silencing is a counterdefensive mechanism that enables systemic infection by TuMV. The suppressor interfered with the activity of miR171 (also known as miRNA39), which directs cleavage of several mRNAs coding for Scarecrow-like transcription factors, by inhibiting miR171-guided nucleolytic function. Out of ten other mRNAs that were validated as miRNA-guided cleavage targets, eight accumulated to elevated levels in the presence of P1/HC-Pro. The basis for TuMV- and other virus-induced disease in plants may be explained, at least partly, by interference with miRNA-controlled developmental pathways that share components with the antiviral RNA-silencing pathway.

MicroRNAs (miRNAs) are approximately 21-nucleotide noncoding RNAs that regulate target transcripts in plants and animals. In addition to miRNAs, plants contain several classes of endogenous small interfering RNAs (siRNAs) involved in target gene regulation and epigenetic silencing. Small RNA libraries were constructed from wild-type Arabidopsis (Arabidopsis thaliana) and mutant plants (rdr2 and dcl3) that were genetically enriched for miRNAs, and a computational procedure was developed to identify candidate miRNAs. Thirty-eight distinct miRNAs corresponding to 22 families were represented in the libraries. Using a 5# rapid amplification of cDNA ends procedure, the transcription start sites for 63 miRNA primary transcripts from 52 MIRNA loci (99 loci tested) were mapped, revealing features consistent with an RNA polymerase II mechanism of transcription. Ten loci (19%) yielded transcripts from multiple start sites. A canonical TATA box motif was identified upstream of the major start site at 45 (86%) of the mapped MIRNA loci. The 5#-mapping data were combined with miRNA cloning and 3#-PCR data to definitively validate expression of at least 73 MIRNA genes. These data provide a molecular basis to explore regulatory mechanisms of miRNA expression in plants. MicroRNAs (miRNAs) are approximately 21-nt noncoding RNAs that posttranscriptionally regulate expression of target genes in plants and animals (Bartel, 2004). Mature miRNAs are generated through multiple processing steps from primary transcripts (pri-miRNA) that contain imperfect foldback structures. In animals, MIRNA genes are transcribed by RNA polymerase II (pol II; Bracht et al., 2004;Cai et al., 2004;Lee et al., 2004), yielding a pri-miRNA that is processed initially by the nuclear RNaseIII-like enzyme Drosha (Lee et al., 2003). The resulting premiRNA transcripts are transported to the cytoplasm and processed by Dicer to yield mature miRNAs (Lee et al., 2002). Less is known about the miRNA biogenesis pathway in plants, although most or all miRNAs require Dicer-like1 (DCL1;Park et al., 2002;Reinhart et al., 2002). The lack of a Drosha ortholog in plants and the finding that DCL1 functions at multiple steps during biogenesis of miR163 suggest that plant miRNA biogenesis may differ somewhat from animals (Kurihara and Watanabe, 2004). miRNAs in both animals and plants incorporate into an effector complex known as the RNA-induced silencing complex and guide either translation-associated repression or cleavage of target mRNAs (Bartel, 2004).Computational and molecular cloning strategies revealed nearly 100 potential MIRNA genes in the Arabidopsis (Arabidopsis thaliana) genome (Llave et al., 2002a;Mette et al., 2002;Park et al., 2002;Reinhart et al., 2002;Palatnik et al., 2003;Jones-Rhoades and Bartel, 2004;Sunkar and Zhu, 2004;Wang et al., 2004). These miRNAs target mRNAs encoding proteins that include a variety of transcription factors involved in development, miRNA/small interfering RNA (siR-NA) metabolic or effector components (DCL1, Argonaute1 [AGO1], and AGO...

MicroRNAs (miRNAs) in plants and animals function as post-transcriptional regulators of target genes, many of which are involved in multicellular development. miRNAs guide effector complexes to target mRNAs through base-pair complementarity, facilitating site-specific cleavage or translational repression. Biogenesis of miRNAs involves nucleolytic processing of a precursor transcript with extensive foldback structure. Here, we provide evidence that genes encoding miRNAs in plants originated by inverted duplication of target gene sequences. Several recently evolved genes encoding miRNAs in Arabidopsis thaliana and other small RNA-generating loci possess the hallmarks of inverted duplication events that formed the arms on each side of their respective foldback precursors. We propose a model for miRNA evolution that suggests a mechanism for de novo generation of new miRNA genes with unique target specificities.

Arabidopsis thaliana contains four DICER-LIKE (DCL) genes with specialized functions in small RNA biogenesis for RNA interferencerelated processes. A mutant with defects in DCL4 was identified and analyzed for microRNA-and endogenous, small interfering RNA (siRNA)-related functions. The dcl4-2 mutant contained normal or near-normal levels of microRNAs (21 nt) and heterochromatin-associated siRNAs (24 nt). In contrast, this mutant lacked each of three families of 21-nt trans-acting siRNAs (ta-siRNAs) and possessed elevated levels of ta-siRNA target transcripts. The dcl4-2 mutant resembled an rna-dependent RNA polymerase 6 mutant in that both mutants lacked ta-siRNAs and displayed heterochronic defects in which vegetative phase change was accelerated. Double mutant analyses with dcl2-1, dcl3-1, and dcl4-2 alleles revealed hierarchical redundancy among DCL activities, leading to alternative processing of ta-siRNA precursors in the absence of DCL4. These data support the concept that plants have specialized and compartmentalized DCL functions for biogenesis of distinct small RNA classes.

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.