Edwards Allen

Control of leaf morphogenesis by microRNAs

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.

P1/HC-Pro, a Viral Suppressor of RNA Silencing, Interferes with Arabidopsis Development and miRNA Function

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.

Evolution of microRNA genes by inverted duplication of target gene sequences in Arabidopsis thaliana.

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.

Expression of Arabidopsis MIRNA Genes

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.

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.

Sequence and Expression Differences Underlie Functional Specialization of Arabidopsis MicroRNAs miR159 and miR319

Many microRNAs (miRNAs) are encoded by small gene families. In a third of all conserved Arabidopsis miRNA families, members vary at two or more nucleotide positions. We have focused on the related miR159 and miR319 families, which share sequence identity at 17 of 21 nucleotides, yet affect different developmental processes through distinct targets. MiR159 regulates MYB mRNAs, while miR319 predominantly acts on TCP mRNAs. In the case of miR319, MYB targeting plays at most a minor role because miR319 expression levels and domain limit its ability to affect MYB mRNAs. In contrast, in the case of miR159, the miRNA sequence prevents effective TCP targeting. We complement these observations by identifying nucleotide positions relevant for miRNA activity with mutants recovered from a suppressor screen. Together, our findings reveal that functional specialization of miR159 and miR319 is achieved through both expression and sequence differences.

Identification and characterization of human cytomegalovirus-encoded microRNAs

ABSTRACT MicroRNAs (miRNAs) are an extensive class of noncoding genes that regulate gene expression through posttranscriptional repression. Given the potential for large viral genomes to encode these transcripts, we examined the human cytomegalovirus AD169 genome for miRNAs using a bioinformatics approach. We identified 406 potential stem-loops, of which 110 were conserved between chimpanzee cytomegalovirus and several strains of human cytomegalovirus. Of these conserved stem-loops, 13 exhibited a significant score using the MiRscan algorithm. Examination of total RNA from human cytomegalovirus-infected cells demonstrated that 5 of the 13 predicted miRNAs were expressed during infection. These studies demonstrate that human cytomegalovirus encodes multiple conserved miRNAs and suggest that human cytomegalovirus may utilize an miRNA strategy to regulate cellular and viral gene function.

ASRP: the Arabidopsis Small RNA Project Database.

Eukaryotes produce functionally diverse classes of small RNAs (20–25 nt). These include microRNAs (miRNAs), which act as regulatory factors during growth and development, and short-interfering RNAs (siRNAs), which function in several epigenetic and post-transcriptional silencing systems. The Arabidopsis Small RNA Project (ASRP) seeks to characterize and functionally analyze the major classes of endogenous small RNAs in plants. The ASRP database provides a repository for sequences of small RNAs cloned from various Arabidopsis genotypes and tissues. Version 3.0 of the database contains 1920 unique sequences, with tools to assist in miRNA and siRNA identification and analysis. The comprehensive database is publicly available through a web interface at http://asrp.cgrb.oregonstate.edu.

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.

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.