Identification And Characterization Of IYO And QQT2, Two Novel Components In Arabidopsis RdDM Pathway

DNA methylation is an important epigenetic mark that regulates gene expression and chromatin state. In plants, DNA methylation can be established through RNA-directed DNA methylation (RdDM) pathway. The RdDM pathway involves two plant specific RNA polymerases (Pols) Ⅳ and Ⅴ, which are evolved from the canonical RNA polymerase Pol Ⅱ. Pol Ⅳ recognizes heterochromatic regions and transcribes precursor RNAs that are copied into long dsRNAs by the RNA-dependent-RNA polymerase RDR2, and processed into 24-nt heterochromatic small interfering RNAs (hc-siRNAs) by DCL3 (Dicer-like 3). The hc-siRNAs were then incorporated into ARGONAUTE4 (AG04) in cytoplasm. The AG04/siRNA complexes are re-imported to the nucleus, where they base pair with nascent scaffold transcripts from Pol Ⅴ and recruit DRM2 (Domains Rearranged Methyltransferase2) to mediate DNA methylation at RdDM loci.In order to identify new components of RdDM pathway, we carried out a genetic screen based on the expression of AGO4, which will be destabilized when hc-siRNAs are reduced. A GFP-AGO4 transgenic line was used for mutagenesis and from the screen, we obtained 68 das(defective in AGO4 stability) mutants, including 65 new alleles of known RdDM components and 3 novel mutants.In this study, we characterized two of the mutants, dasl and das2. The levels of hc-siRNAs and DNA methylation were globally decreased in dasl and das2 mutants, indicating that DAS1 and DAS2 are essential for RdDM pathway. Importantly, those loci affected by dasl and das2 mutations largely overlap with those of nrpdl and nrpe1, mutants of the largest subunits of Pol Ⅳ and Pol Ⅴ. repectively, suggesting that DAS1 and DAS2 might regulate the function of Pol Ⅳ and Pol Ⅴ. Map-based cloning revealed that DAS1 and DAS2 encodes IYO (MINIYO) and QQT2 (QUATRE-QUART2). respectively. The homologs of IYO and QQT2 in yeast and humans were found to be associated with Pol Ⅱ, and Npa3, the homolog of QQT2 in yeast, was recently suggested to participate in Pol Ⅱ assembly. Our biochemical evidence showed that IYO and QQT2 interact with multiple subunits of RNA polymerases, including those shared by Pols Ⅱ, Ⅳ and Ⅴ. Furthermore, we found that the holoenzyme of Pol Ⅴ was incomplete and the largest subunit NRPE1 was dissociated from other subunits in both das1/iyo and das2/qqt2 mutants. In addition, das1/iyo and das2/qqt2 mutations led to reduced protein level and nuclear localization of NRPE1, further supporting the roles of IYO and QQT2 in Pol Ⅴ assembly. Our work thus uncovered the mechanism of two novel components in regulating RdDM and revealed a new layer in the regulation of RdDM pathway.Pol Ⅴ is homologous to Pols Ⅳ and Ⅱ, and they share multiple subunits. Future studies on the mechanisms of IYO and QQT2 in Pol Ⅴ assembly will shed light on our understanding of the assembly of other plant polymerases as well as polymerases in other eukaryotic species.