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An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation.

Gao Z, Liu HL, Daxinger L, Pontes O, He X, Qian W, Lin H, Xie M, Lorkovic ZJ, Zhang S, Miki D, Zhan X, Pontier D, Lagrange T, Jin H, Matzke AJ, Matzke M, Pikaard CS, Zhu JK - Nature (2010)

Bottom Line: Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nucleotide siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci.Our results indicate that RDM1 is a component of the RdDM effector complex and may have a role in linking siRNA production with pre-existing or de novo cytosine methylation.Our results also indicate that, although RDM1 and Pol V (also known as NRPE) may function together at some RdDM target sites in the peri-nucleolar siRNA processing centre, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrative Genome Biology and Department of Botany and Plant Sciences, University of California, Riverside, California 92521, USA.

ABSTRACT
DNA methylation is an important epigenetic mark in many eukaryotes. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2, 4-6). Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nucleotide siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that seems to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II (Pol II, also known as NRPB), AGO4 and DRM2 in the nucleus. Our results indicate that RDM1 is a component of the RdDM effector complex and may have a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also indicate that, although RDM1 and Pol V (also known as NRPE) may function together at some RdDM target sites in the peri-nucleolar siRNA processing centre, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm.

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Sub-nuclear localization of RDM1 and its co-localization with other components of the RdDM pathwaya, The nuclear distribution of RDM1 was analyzed by immunostaining using anti-RDM1 (green). b, Dual immunolocalization using anti-RDM1 (green) in transgenic lines expressing recombinant full-length epitope tagged FLAG-NRPD1, Myc-AGO4, FLAG-NRPE1 and FLAG-DRM2 (red). c, Dual immunolocalization using anti-NRPB1 and anti-RDM1 or anti-AGO4. In all panels the DNA was stained with DAPI (blue) and the size bars correspond to 5 μm. Arrows point to the peri-nucleolar dot (siRNA processing center).
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Figure 4: Sub-nuclear localization of RDM1 and its co-localization with other components of the RdDM pathwaya, The nuclear distribution of RDM1 was analyzed by immunostaining using anti-RDM1 (green). b, Dual immunolocalization using anti-RDM1 (green) in transgenic lines expressing recombinant full-length epitope tagged FLAG-NRPD1, Myc-AGO4, FLAG-NRPE1 and FLAG-DRM2 (red). c, Dual immunolocalization using anti-NRPB1 and anti-RDM1 or anti-AGO4. In all panels the DNA was stained with DAPI (blue) and the size bars correspond to 5 μm. Arrows point to the peri-nucleolar dot (siRNA processing center).

Mentions: Immunostaining of RDM1 yielded fluorescence signals dispersed throughout the nucleoplasm without any preferential accumulation near the DAPI-intensive chromocenters. A prominent signal was observed at the peri-nucleolar siRNA processing center13-15 (Figure 4A). The intensity of the immunosignals was considerably weaker in the rdm1-1 mutant (Figure 4A, bottom row), consistent with the reduced RDM1 protein level in the mutant (Figure 3A). To ascertain whether RDM1 may be co-localized with other components of the RdDM pathway, we performed double immunolocalization using the antibody specific to RDM1 in interphase nuclei from Arabidopsis transgenic lines expressing FLAG-tagged NRPD1, NRPE113 or DRM2, or Myc-tagged AGO414. Yellow signals occurring where red (NRPD1, NRPE1, AGO4 or DRM2) and green (RDM1) signals overlap reveal that RDM1 significantly overlaps with AGO4 and DRM2 in the nucleus (Figure 4B), in agreement with the co-immunoprecipitation between the proteins. The strong peri-nucleolar signals of NRPE1 and RDM1 also overlap, but little overlap between the two proteins is observed in the nucleoplasm. There is also negligible overlap between RDM1 and NRPD1 signals (Figure 4B). In accordance with the co-immunoprecipitation between RDM1 and NRPB1 of Pol II, we found a substantial overlap between the two proteins in the nucleoplasm (Figure 4C). Furthermore, there is also a substantial overlap between AGO4 and NRPB1 signals in the nucleoplasm (Figure 4C). We found that the AGO4 interphase pattern is altered in the rdm1-1 mutant (Figure S8 and Table S1), suggesting that AGO4 localization is dependent on RDM1. By contrast, NRPD1 and NRPE1 maintained their typical interphase localization patterns in the rdm1-1 mutant (Figure S8).


An RNA polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation.

Gao Z, Liu HL, Daxinger L, Pontes O, He X, Qian W, Lin H, Xie M, Lorkovic ZJ, Zhang S, Miki D, Zhan X, Pontier D, Lagrange T, Jin H, Matzke AJ, Matzke M, Pikaard CS, Zhu JK - Nature (2010)

Sub-nuclear localization of RDM1 and its co-localization with other components of the RdDM pathwaya, The nuclear distribution of RDM1 was analyzed by immunostaining using anti-RDM1 (green). b, Dual immunolocalization using anti-RDM1 (green) in transgenic lines expressing recombinant full-length epitope tagged FLAG-NRPD1, Myc-AGO4, FLAG-NRPE1 and FLAG-DRM2 (red). c, Dual immunolocalization using anti-NRPB1 and anti-RDM1 or anti-AGO4. In all panels the DNA was stained with DAPI (blue) and the size bars correspond to 5 μm. Arrows point to the peri-nucleolar dot (siRNA processing center).
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Figure 4: Sub-nuclear localization of RDM1 and its co-localization with other components of the RdDM pathwaya, The nuclear distribution of RDM1 was analyzed by immunostaining using anti-RDM1 (green). b, Dual immunolocalization using anti-RDM1 (green) in transgenic lines expressing recombinant full-length epitope tagged FLAG-NRPD1, Myc-AGO4, FLAG-NRPE1 and FLAG-DRM2 (red). c, Dual immunolocalization using anti-NRPB1 and anti-RDM1 or anti-AGO4. In all panels the DNA was stained with DAPI (blue) and the size bars correspond to 5 μm. Arrows point to the peri-nucleolar dot (siRNA processing center).
Mentions: Immunostaining of RDM1 yielded fluorescence signals dispersed throughout the nucleoplasm without any preferential accumulation near the DAPI-intensive chromocenters. A prominent signal was observed at the peri-nucleolar siRNA processing center13-15 (Figure 4A). The intensity of the immunosignals was considerably weaker in the rdm1-1 mutant (Figure 4A, bottom row), consistent with the reduced RDM1 protein level in the mutant (Figure 3A). To ascertain whether RDM1 may be co-localized with other components of the RdDM pathway, we performed double immunolocalization using the antibody specific to RDM1 in interphase nuclei from Arabidopsis transgenic lines expressing FLAG-tagged NRPD1, NRPE113 or DRM2, or Myc-tagged AGO414. Yellow signals occurring where red (NRPD1, NRPE1, AGO4 or DRM2) and green (RDM1) signals overlap reveal that RDM1 significantly overlaps with AGO4 and DRM2 in the nucleus (Figure 4B), in agreement with the co-immunoprecipitation between the proteins. The strong peri-nucleolar signals of NRPE1 and RDM1 also overlap, but little overlap between the two proteins is observed in the nucleoplasm. There is also negligible overlap between RDM1 and NRPD1 signals (Figure 4B). In accordance with the co-immunoprecipitation between RDM1 and NRPB1 of Pol II, we found a substantial overlap between the two proteins in the nucleoplasm (Figure 4C). Furthermore, there is also a substantial overlap between AGO4 and NRPB1 signals in the nucleoplasm (Figure 4C). We found that the AGO4 interphase pattern is altered in the rdm1-1 mutant (Figure S8 and Table S1), suggesting that AGO4 localization is dependent on RDM1. By contrast, NRPD1 and NRPE1 maintained their typical interphase localization patterns in the rdm1-1 mutant (Figure S8).

Bottom Line: Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nucleotide siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci.Our results indicate that RDM1 is a component of the RdDM effector complex and may have a role in linking siRNA production with pre-existing or de novo cytosine methylation.Our results also indicate that, although RDM1 and Pol V (also known as NRPE) may function together at some RdDM target sites in the peri-nucleolar siRNA processing centre, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrative Genome Biology and Department of Botany and Plant Sciences, University of California, Riverside, California 92521, USA.

ABSTRACT
DNA methylation is an important epigenetic mark in many eukaryotes. In plants, 24-nucleotide small interfering RNAs (siRNAs) bound to the effector protein, Argonaute 4 (AGO4), can direct de novo DNA methylation by the methyltransferase DRM2 (refs 2, 4-6). Here we report a new regulator of RNA-directed DNA methylation (RdDM) in Arabidopsis: RDM1. Loss-of-function mutations in the RDM1 gene impair the accumulation of 24-nucleotide siRNAs, reduce DNA methylation, and release transcriptional gene silencing at RdDM target loci. RDM1 encodes a small protein that seems to bind single-stranded methyl DNA, and associates and co-localizes with RNA polymerase II (Pol II, also known as NRPB), AGO4 and DRM2 in the nucleus. Our results indicate that RDM1 is a component of the RdDM effector complex and may have a role in linking siRNA production with pre-existing or de novo cytosine methylation. Our results also indicate that, although RDM1 and Pol V (also known as NRPE) may function together at some RdDM target sites in the peri-nucleolar siRNA processing centre, Pol II rather than Pol V is associated with the RdDM effector complex at target sites in the nucleoplasm.

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