19 - LETTERS 2005 Nature Publishing Group...

Info iconThis preview shows pages 1–2. Sign up to view the full content.

View Full Document Right Arrow Icon
Atypical RNA polymerase subunits required for RNA-directed DNA methylation Tatsuo Kanno 1 , Bruno Huettel 1 , M Florian Mette 1,3 , Werner Aufsatz 1 , Estelle Jaligot 1 , Lucia Daxinger 1 , David P Kreil 2 , Marjori Matzke 1 & Antonius J M Matzke 1 RNA-directed DNA methylation, one of several RNA interference–mediated pathways in the nucleus 1 , has been documented in plants 2,3 and in human cells 4,5 .Desp ite progress in identifying the DNA methyltransferases, histone- modifying enzymes and RNA interference proteins needed for RNA-directed DNA methylation 1 , the mechanism remains incompletely understood. We screened for mutants defective in RNA-directed DNA methylation and silencing of a transgene promoter in Arabidopsis thaliana and identiFed three drd complementation groups 6 . DRD1 is a SN±2-like protein 6 required for RNA-directed de novo methylation. We report here that DRD2 and DRD3 correspond to the second-largest subunit and largest subunit, respectively, of a fourth class of DNA-dependent RNA polymerase (polymerase IV) that is unique to plants. DRD3 is a functionally diversiFed homolog of NRPD1a or SDE4, identiFed in a separate screen for mutants defective in post-transcriptional gene silencing 7,8 . The identical DNA methylation patterns observed in all three drd mutants suggest that DRD proteins cooperate to create a substrate for RNA-directed de novo methylation. In the two-component a ¢ promoter silencing system, a target a ¢ promoter drives expression of a GFP reporter gene in seeds 6 .Th e target a ¢ promoter is silenced and methylated only in the presence of an unlinked silencer complex, which encodes a hairpin RNA contain- ing a ¢ promoter sequences ( Fig. 1 ). In drd2 and drd3 plants, there is heavy loss of non-CG methylation accompanied by residual CG methylation ( Fig. 1 and Supplementary Fig. 1 online). An identical hybridization pattern was observed in drd1 mutants 6 . Although symmetrical CG and CNG methylation can be maintained in some cases in the absence of the RNA trigger 9–11 , the loss of asymmetrical CNN methylation suggests that there are defects in de novo methylation, which needs the continuous presence of RNA 9 . Indeed, DRD1 is required for RNA-directed de novo methylation of target promoters 12 . The lack of non-CG methylation of the target a ¢ promoter in drd2 and drd3 mutants does not reFect insuf±cient production of RNA drd2 leaf T T+S drd2 seed drd3 leaf drd3 seed –HFS P a Ps E B 35S pro α′ promoter α′ promoter α′ promoter Target a b Silencer KP s P a S –257 +1 Probe GFP HF E S B N Figure 1 Non-CG methylation is lost in drd mutants. ( a ) Methylation of the target a ¢ promoter was analyzed by digestion with restriction enzymes sensitive to non-CG methylation ( Fnu 4HI, F; Scr F1, S; Pst I, Ps; Pag I, Pa; Eco T22I, E; Bam HI, B) and CG methylation ( Hpy CH4IV, H), after a double digestion with methylation-insensitive ( À ) Kpn I (K) and Nde I (N). The small arrow at the left indicates the methylated fragment.
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 2
This is the end of the preview. Sign up to access the rest of the document.

This note was uploaded on 08/01/2009 for the course 1 1 taught by Professor Maz during the Spring '01 term at Abilene Christian University.

Page1 / 5

19 - LETTERS 2005 Nature Publishing Group...

This preview shows document pages 1 - 2. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online