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RNA-directed DNA methylation requires stepwise binding of silencing factors to long non-coding RNA.

Böhmdorfer G, Rowley MJ, Kuciński J, Zhu Y, Amies I, Wierzbicki AT - Plant J. (2014)

Bottom Line: We found that the effector protein ARGONAUTE4 (AGO4) binds lncRNA independent of the RNA-binding protein INVOLVED IN DE NOVO2 (IDN2).We further found that the de novo DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) also associates with lncRNA produced by Pol V and that this event depends on AGO4 and IDN2.We propose a model where the silencing proteins AGO4, IDN2 and DRM2 bind to lncRNA in a stepwise manner, resulting in DNA methylation of RdDM target loci.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA.

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Association of DRM2 with Pol V transcripts requires small interfering RNA (siRNA), AGO4 and IDN2. (a–f) RNA immunoprecipitation (RIP) experiments with an α-DRM2 antibody and nuclei isolated from wild type and RNA-directed DNA methylation (RdDM) mutant seedlings. Mean values and standard deviations of at least two biological replicates are shown. Experiments were performed on the following loci: IGN29 (b), IGN34 (c), IGN35 (d), and IGN36 (e). Pol II-transcribed ACTIN2 served as a control for equal total RNA levels (a) and for potential contamination with genomic DNA (f). (g) Western blot with an α-DRM2 antibody and proteins isolated from wild type (wt) and RdDM mutant seedlings. Ponceau staining of the membrane shows comparable loading of protein extracts. The experiment was performed twice and one representative biological replicate is depicted.
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fig04: Association of DRM2 with Pol V transcripts requires small interfering RNA (siRNA), AGO4 and IDN2. (a–f) RNA immunoprecipitation (RIP) experiments with an α-DRM2 antibody and nuclei isolated from wild type and RNA-directed DNA methylation (RdDM) mutant seedlings. Mean values and standard deviations of at least two biological replicates are shown. Experiments were performed on the following loci: IGN29 (b), IGN34 (c), IGN35 (d), and IGN36 (e). Pol II-transcribed ACTIN2 served as a control for equal total RNA levels (a) and for potential contamination with genomic DNA (f). (g) Western blot with an α-DRM2 antibody and proteins isolated from wild type (wt) and RdDM mutant seedlings. Ponceau staining of the membrane shows comparable loading of protein extracts. The experiment was performed twice and one representative biological replicate is depicted.

Mentions: Experiments described above as well as previously published work (He et al., 2009; Wierzbicki et al., 2009; Rowley et al., 2011; Zhu et al., 2013) have demonstrated that three proteins (AGO4, SPT5L and IDN2) found to work downstream of Pol V actually associate with Pol V transcripts. Therefore, we hypothesized that DRM2 may also bind lncRNA produced by Pol V. To test this hypothesis, we performed RIP experiments with an α-DRM2 antibody. We were able to amplify RNA from the Col-0 wild type at all four tested loci, while the signal was undetectable or was strongly reduced in the drm2 mutant (Figure4b–e), indicating that DRM2 associates with RNA at these loci. The signal was also undetectable or strongly reduced in the nrpe1 mutant (Figure4b–e). Therefore, we concluded that that DRM2 associates specifically with Pol V transcripts at these loci.


RNA-directed DNA methylation requires stepwise binding of silencing factors to long non-coding RNA.

Böhmdorfer G, Rowley MJ, Kuciński J, Zhu Y, Amies I, Wierzbicki AT - Plant J. (2014)

Association of DRM2 with Pol V transcripts requires small interfering RNA (siRNA), AGO4 and IDN2. (a–f) RNA immunoprecipitation (RIP) experiments with an α-DRM2 antibody and nuclei isolated from wild type and RNA-directed DNA methylation (RdDM) mutant seedlings. Mean values and standard deviations of at least two biological replicates are shown. Experiments were performed on the following loci: IGN29 (b), IGN34 (c), IGN35 (d), and IGN36 (e). Pol II-transcribed ACTIN2 served as a control for equal total RNA levels (a) and for potential contamination with genomic DNA (f). (g) Western blot with an α-DRM2 antibody and proteins isolated from wild type (wt) and RdDM mutant seedlings. Ponceau staining of the membrane shows comparable loading of protein extracts. The experiment was performed twice and one representative biological replicate is depicted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321213&req=5

fig04: Association of DRM2 with Pol V transcripts requires small interfering RNA (siRNA), AGO4 and IDN2. (a–f) RNA immunoprecipitation (RIP) experiments with an α-DRM2 antibody and nuclei isolated from wild type and RNA-directed DNA methylation (RdDM) mutant seedlings. Mean values and standard deviations of at least two biological replicates are shown. Experiments were performed on the following loci: IGN29 (b), IGN34 (c), IGN35 (d), and IGN36 (e). Pol II-transcribed ACTIN2 served as a control for equal total RNA levels (a) and for potential contamination with genomic DNA (f). (g) Western blot with an α-DRM2 antibody and proteins isolated from wild type (wt) and RdDM mutant seedlings. Ponceau staining of the membrane shows comparable loading of protein extracts. The experiment was performed twice and one representative biological replicate is depicted.
Mentions: Experiments described above as well as previously published work (He et al., 2009; Wierzbicki et al., 2009; Rowley et al., 2011; Zhu et al., 2013) have demonstrated that three proteins (AGO4, SPT5L and IDN2) found to work downstream of Pol V actually associate with Pol V transcripts. Therefore, we hypothesized that DRM2 may also bind lncRNA produced by Pol V. To test this hypothesis, we performed RIP experiments with an α-DRM2 antibody. We were able to amplify RNA from the Col-0 wild type at all four tested loci, while the signal was undetectable or was strongly reduced in the drm2 mutant (Figure4b–e), indicating that DRM2 associates with RNA at these loci. The signal was also undetectable or strongly reduced in the nrpe1 mutant (Figure4b–e). Therefore, we concluded that that DRM2 associates specifically with Pol V transcripts at these loci.

Bottom Line: We found that the effector protein ARGONAUTE4 (AGO4) binds lncRNA independent of the RNA-binding protein INVOLVED IN DE NOVO2 (IDN2).We further found that the de novo DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) also associates with lncRNA produced by Pol V and that this event depends on AGO4 and IDN2.We propose a model where the silencing proteins AGO4, IDN2 and DRM2 bind to lncRNA in a stepwise manner, resulting in DNA methylation of RdDM target loci.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA.

Show MeSH