MicroRNAs and other small RNAs enriched in the Arabidopsis RNA-dependent RNA polymerase-2 mutant.

The Arabidopsis genome contains a highly complex and abundant population of small RNAs, and many of the endogenous siRNAs are dependent on RNA-Dependent RNA Polymerase 2 (RDR2) for their biogenesis. By analyzing an rdr2 loss-of-function mutant using two different parallel sequencing technologies, MPSS and 454, we characterized the complement of miRNAs expressed in Arabidopsis inflorescence to considerable depth. Nearly all known miRNAs were enriched in this mutant and we identified 13 new miRNAs, all of which were relatively low abundance and constitute new families. Trans-acting siRNAs (ta-siRNAs) were even more highly enriched. Computational and gel blot analyses suggested that the minimal number of miRNAs in Arabidopsis is approximately 155. The size profile of small RNAs in rdr2 reflected enrichment of 21-nt miRNAs and other classes of siRNAs like ta-siRNAs, and a significant reduction in 24-nt heterochromatic siRNAs. Other classes of small RNAs were found to be RDR2-independent, particularly those derived from long inverted repeats and a subset of tandem repeats. The small RNA populations in other Arabidopsis small RNA biogenesis mutants were also examined; a dcl2/3/4 triple mutant showed a similar pattern to rdr2, whereas dcl1-7 and rdr6 showed reductions in miRNAs and ta-siRNAs consistent with their activities in the biogenesis of these types of small RNAs. Deep sequencing of mutants provides a genetic approach for the dissection and characterization of diverse small RNA populations and the identification of low abundance miRNAs.

Role of RNA polymerase IV in plant small RNA metabolism.

In addition to the three RNA polymerases (RNAP I-III) shared by all eukaryotic organisms, plant genomes encode a fourth RNAP (RNAP IV) that appears to be specialized in the production of siRNAs. Available data support a model in which dsRNAs are generated by RNAP IV and RNA-dependent RNAP 2 (RDR2) and processed by DICER (DCL) enzymes into 21- to 24-nt siRNAs, which are associated with different ARGONAUTE (AGO) proteins for transcriptional or posttranscriptional gene silencing. However, it is not yet clear what fraction of genomic siRNA production is RNAP IV-dependent, and to what extent these siRNAs are preferentially processed by certain DCL(s) or associated with specific AGOs for distinct downstream functions. To address these questions on a genome-wide scale, we sequenced approximately 335,000 siRNAs from wild-type and RNAP IV mutant Arabidopsis plants by using 454 technology. The results show that RNAP IV is required for the production of >90% of all siRNAs, which are faithfully produced from a discrete set of genomic loci. Comparisons of these siRNAs with those accumulated in rdr2 and dcl2 dcl3 dcl4 and those associated with AGO1 and AGO4 provide important information regarding the processing, channeling, and functions of plant siRNAs. We also describe a class of RNAP IV-independent siRNAs produced from endogenous single-stranded hairpin RNA precursors.

Epigenetic remodeling of meiotic crossover frequency in Arabidopsis thaliana DNA methyltransferase mutants.

Meiosis is a specialized eukaryotic cell division that generates haploid gametes required for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover (CO). Meiotic CO frequency varies along the physical length of chromosomes and is determined by hierarchical mechanisms, including epigenetic organization, for example methylation of the DNA and histones. Here we investigate the role of DNA methylation in determining patterns of CO frequency along Arabidopsis thaliana chromosomes. In A. thaliana the pericentromeric regions are repetitive, densely DNA methylated, and suppressed for both RNA polymerase-II transcription and CO frequency. DNA hypomethylated methyltransferase1 (met1) mutants show transcriptional reactivation of repetitive sequences in the pericentromeres, which we demonstrate is coupled to extensive remodeling of CO frequency. We observe elevated centromere-proximal COs in met1, coincident with pericentromeric decreases and distal increases. Importantly, total numbers of CO events are similar between wild type and met1, suggesting a role for interference and homeostasis in CO remodeling. To understand recombination distributions at a finer scale we generated CO frequency maps close to the telomere of chromosome 3 in wild type and demonstrate an elevated recombination topology in met1. Using a pollen-typing strategy we have identified an intergenic nucleosome-free CO hotspot 3a, and we demonstrate that it undergoes increased recombination activity in met1. We hypothesize that modulation of 3a activity is caused by CO remodeling driven by elevated centromeric COs. These data demonstrate how regional epigenetic organization can pattern recombination frequency along eukaryotic chromosomes.

An ARGONAUTE4-containing nuclear processing center colocalized with Cajal bodies in Arabidopsis thaliana.

ARGONAUTE4 (AGO4) and RNA polymerase IV (Pol IV) are required for DNA methylation guided by 24 nucleotide small interfering RNAs (siRNAs) in Arabidopsis thaliana. Here we show that AGO4 localizes to nucleolus-associated bodies along with the Pol IV subunit NRPD1b; the small nuclear RNA (snRNA) binding protein SmD3; and two markers of Cajal bodies, trimethylguanosine-capped snRNAs and the U2 snRNA binding protein U2B''. AGO4 interacts with the C-terminal domain of NRPD1b, and AGO4 protein stability depends on upstream factors that synthesize siRNAs. AGO4 is also found, along with the DNA methyltransferase DRM2, throughout the nucleus at presumed DNA methylation target sites. Cajal bodies are conserved sites for the maturation of ribonucleoprotein complexes. Our results suggest a function for Cajal bodies as a center for the assembly of an AGO4/NRPD1b/siRNA complex, facilitating its function in RNA-directed gene silencing at target loci.

Dynamic regulation of ARGONAUTE4 within multiple nuclear bodies in Arabidopsis thaliana.

DNA methylation directed by 24-nucleotide small RNAs involves the small RNA-binding protein ARGONAUTE4 (AGO4), and it was previously shown that AGO4 localizes to nucleolus-adjacent Cajal bodies, sites of snRNP complex maturation. Here we demonstrate that AGO4 also localizes to a second class of nuclear bodies, called AB-bodies, which are found immediately adjacent to condensed 45S ribosomal DNA (rDNA) sequences. AB-bodies also contain other proteins involved in RNA-directed DNA methylation including NRPD1b (a subunit of the RNA Polymerase IV complex, RNA PolIV), NRPD2 (a second subunit of this complex), and the DNA methyltransferase DRM2. These two classes of AGO4 bodies are structurally independent--disruption of one class does not affect the other--suggesting a dynamic regulation of AGO4 within two distinct nuclear compartments in Arabidopsis. Abolishing Cajal body formation in a coilin mutant reduced overall AGO4 protein levels, and coilin dicer-like3 double mutants showed a small decrease in DNA methylation beyond that seen in dicer-like3 single mutants, suggesting that Cajal bodies are required for a fully functioning DNA methylation system in Arabidopsis.