Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2.

RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In Arabidopsis, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest, induced by the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The double-stranded RNAs are then released from the Pol IV-RDR2 complex and diced into short-interfering RNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 Å, determined by cryoelectron microscopy. The N-terminal region contains an RNA-recognition motif adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1 to 2 nt internal to the 3' ends of its templates and can transcribe the RNA of an RNA/DNA hybrid, provided that 9 or more nucleotides are unpaired at the RNA's 3' end. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3' end occurs as the DNA template and nontemplate strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3' end as the DNA strands reanneal, allowing RDR2 to engage the RNA and synthesize the complementary strand.

The REL3-mediated TAS3 ta-siRNA pathway integrates auxin and ethylene signaling to regulate nodulation in Lotus japonicus.

The ta-siRNA pathway is required for lateral organ development, including leaf patterning, flower differentiation and lateral root growth. Legumes can develop novel lateral root organs--nodules--resulting from symbiotic interactions with rhizobia. However, ta-siRNA regulation in nodule formation remains unknown. To explore ta-siRNA regulation in nodule formation, we investigated the roles of REL3, a key component of TAS3 ta-siRNA biogenesis, during nodulation in Lotus japonicus. We characterized the symbiotic phenotypes of the TAS3 ta-siRNA defective rel3 mutant, and analyzed the responses of the rel3 mutant to auxin and ethylene in order to gain insight into TAS3 ta-siRNA regulation of nodulation. The rel3 mutant produced fewer pink nitrogen-fixing nodules, with substantially decreased infection frequency and nodule initiation. Moreover, the rel3 mutant was more resistant than wild-type to 1-naphthaleneacetic acid (NAA) and N-1-naphthylphthalamic acid (NPA) in root growth, and exhibited insensitivity to auxins but greater sensitivity to auxin transport inhibitors during nodulation. Furthermore, the rel3 mutant has enhanced root-specific ethylene sensitivity and altered responses to ethylene during nodulation; the low-nodulating phenotype of the rel3 mutant can be restored by ethylene synthesis inhibitor L-α-(2-aminoethoxyvinyl)-glycine (AVG) or action inhibitor Ag(+). The REL3-mediated TAS3 ta-siRNA pathway regulates nodulation by integrating ethylene and auxin signaling.

Phase separation of SGS3 drives siRNA body formation and promotes endogenous gene silencing.

The generation of small interfering RNA (siRNA) involves many RNA processing components, including SUPPRESSOR OF GENE SILENCING 3 (SGS3), RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), and DICER-LIKE proteins (DCLs). Nonetheless, how these components are coordinated to produce siRNAs is unclear. Here, we show that SGS3 forms condensates via phase separation in vivo and in vitro. SGS3 interacts with RDR6 and drives it to form siRNA bodies in cytoplasm, which is promoted by SGS3-targeted RNAs. Disrupting SGS3 phase separation abrogates siRNA body assembly and siRNA biogenesis, whereas coexpression of SGS3 and RDR6 induces siRNA body formation in tobacco and yeast cells. Dysfunction in translation and mRNA decay increases the number of siRNA bodies, whereas DCL2/4 mutations enhance their size. Purification of SGS3 condensates identifies numerous RNA-binding proteins and siRNA processing components. Together, our findings reveal that SGS3 phase separation-mediated formation of siRNA bodies is essential for siRNA production and gene silencing.

A DCL3 dicing code within Pol IV-RDR2 transcripts diversifies the siRNA pool guiding RNA-directed DNA methylation.

In plants, selfish genetic elements, including retrotransposons and DNA viruses, are transcriptionally silenced by RNA-directed DNA methylation. Guiding the process are short interfering RNAs (siRNAs) cut by DICER-LIKE 3 (DCL3) from double-stranded precursors of ~30 bp that are synthesized by NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). We show that Pol IV's choice of initiating nucleotide, RDR2's initiation 1-2 nt internal to Pol IV transcript ends and RDR2's terminal transferase activity collectively yield a code that influences which precursor end is diced and whether 24 or 23 nt siRNAs are produced. By diversifying the size, sequence, and strand specificity of siRNAs derived from a given precursor, alternative patterns of DCL3 dicing allow for maximal siRNA coverage at methylated target loci.

Modeling of the catalytic core of Arabidopsis thaliana Dicer-like 4 protein and its complex with double-stranded RNA.

Plant Dicer-like proteins (DCLs) belong to the Ribonuclease III (RNase III) enzyme family. They are involved in the regulation of gene expression and antiviral defense through RNA interference pathways. A model plant, Arabidopsis thaliana encodes four DCL proteins (AtDCL1-4) that produce different classes of small regulatory RNAs. Our studies focus on AtDCL4 that processes double-stranded RNAs (dsRNAs) into 21 nucleotide trans-acting small interfering RNAs. So far, little is known about the structures of plant DCLs and the complexes they form with dsRNA. In this work, we present models of the catalytic core of AtDCL4 and AtDCL4-dsRNA complex constructed by computational methods. We built a homology model of the catalytic core of AtDCL4 comprising Platform, PAZ, Connector helix and two RNase III domains. To assemble the AtDCL4-dsRNA complex two modeling approaches were used. In the first method, to establish conformations that allow building a consistent model of the complex, we used Normal Mode Analysis for both dsRNA and AtDCL4. The second strategy involved template-based approach for positioning of the PAZ domain and manual arrangement of the Connector helix. Our results suggest that the spatial orientation of the Connector helix, Platform and PAZ relative to the RNase III domains is crucial for measuring dsRNA of defined length. The modeled complexes provide information about interactions that may contribute to the relative orientations of these domains and to dsRNA binding. All these information can be helpful for understanding the mechanism of AtDCL4-mediated dsRNA recognition and binding, to produce small RNA of specific size.