Reaction Mechanisms of Pol IV, RDR2, and DCL3 Drive RNA Channeling in the siRNA-Directed DNA Methylation Pathway

Mol Cell. 2019 Aug 8;75(3):576-589.e5. doi: 10.1016/j.molcel.2019.07.008.

Abstract

In eukaryotes with multiple small RNA pathways, the mechanisms that channel RNAs within specific pathways are unclear. Here, we reveal the reactions that account for channeling in the small interfering RNA (siRNA) biogenesis phase of the Arabidopsis RNA-directed DNA methylation pathway. The process begins with template DNA transcription by NUCLEAR RNA POLYMERASE IV (Pol IV), whose atypical termination mechanism, induced by nontemplate DNA base-pairing, channels transcripts to the associated RNA-dependent RNA polymerase RDR2. RDR2 converts Pol IV transcripts into double-stranded RNAs and then typically adds an extra untemplated 3' terminal nucleotide to the second strands. The dicer endonuclease DCL3 cuts resulting duplexes to generate 24- and 23-nt siRNAs. The 23-nt RNAs bear the untemplated terminal nucleotide of the RDR2 strand and are underrepresented among ARGONAUTE4-associated siRNAs. Collectively, our results provide mechanistic insights into Pol IV termination, Pol IV-RDR2 coupling, and RNA channeling, from template DNA transcription to siRNA strand discrimination.

Keywords: DICER-LIKE 3; RNA silencing; RNA-DEPENDENT RNA POLYMERASE 2; RNA-directed DNA methylation; dicing; dsRNA synthesis; ncRNA processing; noncoding RNA; nuclear RNA polymerase IV; transcription termination.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / genetics
  • Arabidopsis Proteins / genetics*
  • Argonaute Proteins / genetics
  • DNA Methylation / genetics
  • DNA, Plant / genetics
  • DNA-Directed RNA Polymerases / genetics*
  • Gene Expression Regulation, Plant / genetics
  • Gene Silencing
  • RNA, Double-Stranded / genetics
  • RNA, Small Interfering / genetics
  • RNA-Dependent RNA Polymerase / genetics*
  • Ribonuclease III / genetics*
  • Signal Transduction
  • Transcription, Genetic*

Substances

  • AGO4 protein, Arabidopsis
  • Arabidopsis Proteins
  • Argonaute Proteins
  • DNA, Plant
  • RNA, Double-Stranded
  • RNA, Small Interfering
  • RNA polymerase IV, Arabidopsis
  • RDR2 protein, Arabidopsis
  • RNA-Dependent RNA Polymerase
  • DNA-Directed RNA Polymerases
  • DCL3 protein, Arabidopsis
  • Ribonuclease III