RNA-directed DNA methylation requires stepwise binding of silencing factors to long non-coding RNA.
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.
Affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA.Show MeSH
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.
Affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA.