INVOLVED IN DE NOVO 2-containing complex involved in RNA-directed DNA methylation in Arabidopsis

Abstract

At least three pathways control maintenance of DNA cytosine methylation in Arabidopsis thaliana. However, the RNA-directed DNA methylation (RdDM) pathway is solely responsible for establishment of this silencing mark. We previously described INVOLVED IN DE NOVO 2 (IDN2) as being an RNA-binding RdDM component that is required for DNA methylation establishment. In this study, we describe the discovery of two partially redundant proteins that are paralogous to IDN2 and that form a stable complex with IDN2 in vivo. Null mutations in both genes, termed IDN2-LIKE 1 and IDN2-LIKE 2 (IDNL1 and IDNL2), result in a phenotype that mirrors, but does not further enhance, the idn2 mutant phenotype. Genetic analysis suggests that this complex acts in a step in the downstream portion of the RdDM pathway. We also have performed structural analysis showing that the IDN2 XS domain adopts an RNA recognition motif (RRM) fold. Finally, genome-wide DNA methylation and expression analysis confirms the placement of the IDN proteins in an RdDM pathway that affects DNA methylation and transcriptional control at many sites in the genome. Results from this study identify and describe two unique components of the RdDM machinery, adding to our understanding of DNA methylation control in the Arabidopsis genome.genomics | mass spectrometry | siRNAs | epigenetics D NA methylation is a stable epigenetic mark that is associated with the repression of genes and transposable elements. In Arabidopsis thaliana, maintenance of DNA methylation at silent loci is carried out by at least three methyltransferases: METH-YLTRANSFERASE 1 (MET1), CHROMOMETHYLTRANS-FERASE 3 (CMT3), and DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2). However, DRM2 is solely responsible for establishment of DNA methylation-or de novo methylation-of silent elements (1). DRM2 is guided to chromatin by small interfering RNAs (siRNAs) in a process known as RNAdirected DNA methylation (RdDM) (2). It has been proposed that RdDM also requires intergenic noncoding (IGN) transcripts that are synthesized by RNA Polymerase V (Pol V). These transcripts likely serve as platforms for the recruitment of siRNA-loaded ARGONAUTE 4 (AGO4) to methylated loci (3, 4).Recently, we discovered the requirement of INVOLVED IN DE NOVO 2 (IDN2) for RdDM from a forward genetic screen (5). Alleles of this same gene were later reported from another screen for DNA methylation mutants (6). We previously demonstrated that IDN2 binds to double-stranded RNA through its XS domain, which is also observed in the XS-domain-containing protein SUPPRESSOR OF GENE SILENCING 3 (SGS3) (7). We also found that IDN2 was likely to act in a step downstream of initial siRNA biogenesis. The XS domain is conserved throughout the plant kingdom, and XS-domain-containing proteins are involved in a wide range of processes such as viral defense (8) and stress response (9).To gain a better understanding of the in vivo role of IDN2, we performed affinity purifications from complementing transgenic lines expressing epitope-tagged full-length I...

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