Identification of Pol IV and RDR2-dependent precursors of 24 nt siRNAs guiding de novo DNA methylation in Arabidopsis

Elife. 2015 Oct 2;4:e09591. doi: 10.7554/eLife.09591.

Abstract

In Arabidopsis thaliana, abundant 24 nucleotide small interfering RNAs (24 nt siRNA) guide the cytosine methylation and silencing of transposons and a subset of genes. 24 nt siRNA biogenesis requires nuclear RNA polymerase IV (Pol IV), RNA-dependent RNA polymerase 2 (RDR2) and DICER-like 3 (DCL3). However, siRNA precursors are mostly undefined. We identified Pol IV and RDR2-dependent RNAs (P4R2 RNAs) that accumulate in dcl3 mutants and are diced into 24 nt RNAs by DCL3 in vitro. P4R2 RNAs are mostly 26-45 nt and initiate with a purine adjacent to a pyrimidine, characteristics shared by Pol IV transcripts generated in vitro. RDR2 terminal transferase activity, also demonstrated in vitro, may account for occasional non-templated nucleotides at P4R2 RNA 3' termini. The 24 nt siRNAs primarily correspond to the 5' or 3' ends of P4R2 RNAs, suggesting a model whereby siRNAs are generated from either end of P4R2 duplexes by single dicing events.

Keywords: RNA interference; RNA silencing; RNA-directed DNA methylation; arabidopsis; chromosomes; epigenetics; genes; noncoding RNA; transcription.

Publication types

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

MeSH terms

  • Arabidopsis / genetics
  • Arabidopsis / physiology*
  • Arabidopsis Proteins / metabolism*
  • DNA Methylation*
  • DNA-Directed RNA Polymerases / metabolism*
  • Gene Expression Regulation, Plant
  • RNA, Small Interfering / metabolism*
  • RNA-Dependent RNA Polymerase / metabolism*
  • Ribonuclease III / deficiency
  • Ribonuclease III / metabolism*

Substances

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