Specific but interdependent functions for Arabidopsis AGO4 and AGO6 in RNA‐directed DNA methylation

Argonaute (AGO) family proteins are conserved key components of small RNA‐induced silencing pathways. In the RNA‐directed DNA methylation (RdDM) pathway in Arabidopsis, AGO6 is generally considered to be redundant with AGO4. In this report, our comprehensive, genomewide analyses of AGO4‐ and AGO6‐dependent DNA methylation revealed that redundancy is unexpectedly negligible in the genetic interactions between AGO4 and AGO6. Immunofluorescence revealed that AGO4 and AGO6 differ in their subnuclear co‐localization with RNA polymerases required for RdDM. Pol II and AGO6 are absent from perinucleolar foci, where Pol V and AGO4 are co‐localized. In the nucleoplasm, AGO4 displays a strong co‐localization with Pol II, whereas AGO6 co‐localizes with Pol V. These patterns suggest that RdDM is mediated by distinct, spatially regulated combinations of AGO proteins and RNA polymerases. Consistently, Pol II physically interacts with AGO4 but not AGO6, and the levels of Pol V‐dependent scaffold RNAs and Pol V chromatin occupancy are strongly correlated with AGO6 but not AGO4. Our results suggest that AGO4 and AGO6 mainly act sequentially in mediating small RNA‐directed DNA methylation.     

Cytoplasmic assembly and selective nuclear import of Arabidopsis Argonaute4/siRNA complexes

In plants, DNA methylation can be mediated by a class of Argonaute4 (AGO4)-associated heterochromatic siRNAs (hc-siRNAs), through a pathway termed RNA-directed DNA methylation (RdDM). It has been thought that RdDM is solely a nuclear process, as both the biogenesis and functioning of hc-siRNAs take place in the nucleus. In this study, we unexpectedly found that hc-siRNAs are predominantly present in the cytoplasm. We demonstrated that AGO4 is loaded with hc-siRNAs in the cytoplasm and the formation of mature AGO4/siRNA complexes requires HSP90 and the cleavage activity of AGO4. Intriguingly, siRNA binding facilitates the redistribution of AGO4 into the nucleus, likely through inducing conformational change that leads to the exposure of the nuclear localization signal (NLS). Our findings reveal an unsuspected cytoplasmic step in the RdDM pathway. We propose that selective nuclear import of mature AGO4/siRNA complexes is a key regulatory point prior to the effector stage of RdDM.     

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.     

Double-stranded RNA binding proteins DRB2 and DRB4 have an antagonistic impact on polymerase IV-dependent siRNA levels in Arabidopsis

Biogenesis of the vast majority of plant siRNAs depends on the activity of the plant-specific RNA polymerase IV (PolIV) enzyme. As part of the RNA-dependent DNA methylation (RdDM) process, PolIV-dependent siRNAs (p4-siRNAs) are loaded onto an ARGONAUTE4-containing complex and guide de novo DNA methyltransferases to target loci. Here we show that the double-stranded RNA binding proteins DRB2 and DRB4 are required for proper accumulation of p4-siRNAs. In flowers, loss of DRB2 results in increased accumulation of p4-siRNAs but not ta-siRNAs, inverted repeat (IR)-derived siRNAs, or miRNA. Loss of DRB2 does not impair uniparental expression of p4-dependent siRNAs in developing endosperm, indicating that p4-siRNA increased accumulation is not the result of the activation of the polIV pathway in the male gametophyte. In contrast to drb2, drb4 mutants exhibit reduced p4-siRNA levels, but the extent of this reduction is variable, according to the nature and size of the p4-siRNAs. Loss of DRB4 also leads to a spectacular increase of p4-independent IR-derived 24-nt siRNAs, suggesting a reallocation of factors from p4-dependent to p4-independent siRNA pathways in drb4. Opposite effects of drb2 and drb4 mutations on the accumulation of p4-siRNAs were also observed in vegetative tissues. Moreover, transgenic plants overexpressing DRB2 mimicked drb4 mutants at the morphological and molecular levels, confirming the antagonistic roles of DRB2 and DRB4.     

RNA‐directed DNA methylation requires an AGO4‐interacting member of the SPT5 elongation factor family

Recent studies have identified a conserved WG/GW‐containing motif, known as the Argonaute (AGO) hook, which is involved in the recruitment of AGOs to distinct components of the eukaryotic RNA silencing pathways. By using this motif as a model to detect new components in plant RNA silencing pathways, we identified SPT5‐like, a plant‐specific AGO4‐interacting member of the nuclear SPT5 (Suppressor of Ty insertion 5) RNA polymerase (RNAP) elongation factor family that is characterized by the presence of a carboxy‐terminal extension with more than 40 WG/GW motifs. Knockout SPT5‐like mutants show a decrease in the accumulation of several 24‐nt RNAs and hypomethylation at different loci revealing an implication in RNA‐directed DNA methylation (RdDM). Here, we propose that SPT5‐like emerged in plants as a facultative RNAP elongation factor. Its plant‐specific origin and role in RdDM might reflect functional interactions with plant‐specific RNA Pols required for RdDM.