The ability to form homodimers is essential for RDM1 to function in RNA-directed DNA methylation.
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.
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan ; Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna, Austria.
Taku
T
Sasaki
Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna, Austria.
Zdravko J
ZJ
Lorković
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan.
Shih-Chieh
SC
Liang
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan.
Antonius J M
AJ
Matzke
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan.
Marjori
M
Matzke
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D060565:Argonaute Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D018744:DNA, Plant / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000037:antagonists & inhibitors / Q000235:genetics*; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction
C471209:AGO4 protein, Arabidopsis; D029681:Arabidopsis Proteins; D060565:Argonaute Proteins; D002868:Chromosomal Proteins, Non-Histone; C529025:DMS3 protein, Arabidopsis; D018744:DNA, Plant; D004268:DNA-Binding Proteins; C549271:RDM1 protein, Arabidopsis; D012333:RNA, Messenger; D018749:RNA, Plant; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D012319:RNA Polymerase II; C538841:RNA polymerase V, Arabidopsis; D012321:DNA-Directed RNA Polymerases