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: As we wanted to understand the order of events downstream of Pol V production, we focused our study on IDN2/IDNL1/IDNL2-dependent loci and asked if lncRNA produced by Pol V at representative loci is bound by AGO4, IDN2 and DRM2. We also determined if these proteins bind to lncRNA in a particular order. We first tested if IDN2 is required for recruiting AGO4 to RdDM targets using RIP with an α-AGO4 antibody. While the experiments revealed that IDN2 is not essential for binding of lncRNA by AGO4, we confirmed that siRNA produced by RDR2 seems to be important for this event, as previously suggested (Wierzbicki et al., 2009; Zheng et al., 2013). Therefore, AGO4 binds to lncRNA independently of IDN2 (Figure6).
Affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, 48109, USA.