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

Abstract

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.

Enzymatic reactions of AGO4 in RNA-directed DNA methylation: siRNA duplex loading, passenger strand elimination, target RNA slicing and sliced target retention

“…24 nt RNAs outnumber 23 nt RNAs by approximately 3:1 in the pool of RNAs associated with slicing-defective AGO4 (Figure 2A), suggesting that 24/24 nt and 24/23 nt duplexes are loaded into AGO4 in similar abundance. Computational analysis of how 24 nt siRNAs can best pair with other, complementary 24 nt siRNAs among the pool of RNAs associated with AGO4-SD, predicts duplexes with 2 nt 3’ overhangs at each end as the most abundant duplex species (Figure S3C, left panel), which matches the known pairing of 24/24 duplexes generated by DCL3 dicing in vitro (Fukudome et al ., 2021; Loffer et al ., 2022). No such pattern was found for 24 nt RNAs associated with wild-type AGO4 (Figure S3C, right panel), consistent with the interpretation that slicing eliminates one strand of any 24/24 duplex that is loaded.…”

“…RNA-directed DNA methylation (RdDM) in Arabidopsis is a TGS pathway in which siRNA biogenesis occurs independent of AGO activity (Matzke and Mosher, 2014; Wendte and Pikaard, 2017; Zhang et al, 2018; Zhou and Law, 2015). Precursors of RdDM siRNAs are synthesized by DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) (Haag and Pikaard, 2011; Ream et al, 2009) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) (Du et al, 2022; Fukudome et al, 2021; Zhou and Law, 2015). These enzymes physically associate (Haag et al, 2012) and carry out coupled reactions that transcribe target locus DNA sequences into double-stranded RNAs of ~25-40bp (Fukudome et al ., 2021; Huang et al, 2021; Singh et al, 2019).…”

“…Precursors of RdDM siRNAs are synthesized by DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) (Haag and Pikaard, 2011; Ream et al, 2009) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) (Du et al, 2022; Fukudome et al, 2021; Zhou and Law, 2015). These enzymes physically associate (Haag et al, 2012) and carry out coupled reactions that transcribe target locus DNA sequences into double-stranded RNAs of ~25-40bp (Fukudome et al ., 2021; Huang et al, 2021; Singh et al, 2019). DICER-LIKE 3 (DCL3) then cuts the dsRNAs into siRNAs, guided by sequence and structural features at the ends of precursor duplexes (Loffer et al, 2022).…”

Structural insights into the dual activities of the two-barrel RNA polymerase QDE-1

“…Unlike At RDR2, Zm RDR2 or At RDR6, which can function as monomers ( Supplementary Figure S11C ; Supplementary Table S5 ) ( 58 , 59 ), QDE-1ΔN exists as a homodimer (Figure 1B - D ; Supplementary Figure S2 ). The C-tail observed in NTP loading and the two initiation states comes from the dimer partner.…”

Enzymatic reactions of AGO4 in RNA-directed DNA methylation: siRNA duplex loading, passenger strand elimination, target RNA slicing, and sliced target retention

“…RNA-directed DNA methylation (RdDM) is the major transcriptional gene silencing pathway in plants ( Matzke and Mosher 2014 ; Zhou and Law 2015 ; Wendte and Pikaard 2017 ; Zhang et al 2018 ). RdDM siRNA precursors are synthesized by DNA-dependent nuclear RNA polymerase IV (Pol IV) ( Ream et al 2009 ; Haag and Pikaard 2011 ) and RNA-dependent RNA polymerase 2 (RDR2) ( Zhou and Law 2015 ; Fukudome et al 2021 ; Du et al 2022 ). These enzymes physically associate and carry out coupled reactions that transcribe target locus DNA sequences into double-stranded RNAs of ∼25–40 bp ( Haag et al 2012 ; Singh et al 2019 ; Fukudome et al 2021 ; Huang et al 2021 ).…”