The ability to form homodimers is essential for RDM1 to function in RNA-directed DNA methylation

PLoS One. 2014 Feb 3;9(2):e88190. doi: 10.1371/journal.pone.0088190. eCollection 2014.

Abstract

RDM1 (RNA-DIRECTED DNA METHYLATION1) is a small plant-specific protein required for RNA-directed DNA methylation (RdDM). RDM1 interacts with RNA polymerase II (Pol II), ARGONAUTE4 (AGO4), and the de novo DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) and binds to methylated single stranded DNA. As the only protein identified so far that interacts directly with DRM2, RDM1 plays a pivotal role in the RdDM mechanism by linking the de novo DNA methyltransferase activity to AGO4, which binds short interfering RNAs (siRNAs) that presumably base-pair with Pol II or Pol V scaffold transcripts synthesized at target loci. RDM1 also acts together with the chromatin remodeler DEFECTIVE IN RNA-DIRECTED DNA METHYLATION1 (DRD1) and the structural-maintenance-of-chromosomes solo hinge protein DEFECTIVE IN MERISTEM SILENCING3 (DMS3) to form the DDR complex, which facilitates synthesis of Pol V scaffold transcripts. The manner in which RDM1 acts in both the DDR complex and as a factor bridging DRM2 and AGO4 remains unclear. RDM1 contains no known protein domains but a prior structural analysis suggested distinct regions that create a hydrophobic pocket and promote homodimer formation, respectively. We have tested several mutated forms of RDM1 altered in the predicted pocket and dimerization regions for their ability to complement defects in RdDM and transcriptional gene silencing, support synthesis of Pol V transcripts, form homodimers, and interact with DMS3. Our results indicate that the ability to form homodimers is essential for RDM1 to function fully in the RdDM pathway and may be particularly important during the de novo methylation step.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / enzymology*
  • Arabidopsis / genetics
  • Arabidopsis / growth & development
  • Arabidopsis Proteins / antagonists & inhibitors
  • Arabidopsis Proteins / genetics*
  • Arabidopsis Proteins / metabolism*
  • Argonaute Proteins / genetics
  • Argonaute Proteins / metabolism*
  • Blotting, Western
  • Chromosomal Proteins, Non-Histone / genetics
  • Chromosomal Proteins, Non-Histone / metabolism*
  • DNA Methylation*
  • DNA, Plant / genetics
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • DNA-Directed RNA Polymerases / antagonists & inhibitors
  • DNA-Directed RNA Polymerases / genetics*
  • Gene Silencing
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Protein Multimerization
  • RNA Polymerase II / antagonists & inhibitors
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism*
  • RNA, Messenger / genetics
  • RNA, Plant / genetics
  • RNA, Small Interfering / genetics
  • Real-Time Polymerase Chain Reaction
  • Reverse Transcriptase Polymerase Chain Reaction

Substances

  • AGO4 protein, Arabidopsis
  • Arabidopsis Proteins
  • Argonaute Proteins
  • Chromosomal Proteins, Non-Histone
  • DMS3 protein, Arabidopsis
  • DNA, Plant
  • DNA-Binding Proteins
  • RDM1 protein, Arabidopsis
  • RNA, Messenger
  • RNA, Plant
  • RNA, Small Interfering
  • Green Fluorescent Proteins
  • RNA Polymerase II
  • RNA polymerase V, Arabidopsis
  • DNA-Directed RNA Polymerases

Grants and funding

The authors thank the Gregor Mendel Institute, Austrian Academy of Sciences, and Academia Sinica for financial support. TS is supported by the Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellowship for Research Abroad. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.