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

Akihito Fukudome, Jasleen Singh, Vibhor Mishra, Eswar Reddem, Francisco Martinez-Marquez, Sabine Wenzel, Rui Yan, Momoko Shiozaki, Zhiheng Yu, Joseph Che Yen Wang, Yuichiro Takagi, Craig S. Pikaard

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

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 30 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 30 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 30 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 30 end as the DNA strands reanneal, allowing RDR2 to engage the RNA and synthesize the complementary strand.

Original languageEnglish (US)
Article numbere2115899118
JournalProceedings of the National Academy of Sciences of the United States of America
Volume118
Issue number51
DOIs
StatePublished - Dec 21 2021

All Science Journal Classification (ASJC) codes

  • General

Fingerprint

Dive into the research topics of 'Structure and RNA template requirements of Arabidopsis RNA-DEPENDENT RNA POLYMERASE 2'. Together they form a unique fingerprint.

Cite this