Cytoplasmic Assembly Of AGO4/siRNA Complexes And DNA Methylation Guided By DCL-independent SRNAs In Arabidopsis

DNA methylation is an important epigenetic mark that regulates gene expression and chromatin state. In plants, DNA methylation can be mediated by a class of Argonaute4(AGO4)-associated heterochromatic small interfereing RNAs (hc-siRNAs), through a pathway termed RNA-directed DNA methylation (RdDM). It has been thought that RdDM is solely a nuclear process as both the biogenesis and functioning of hc-siRNAs take place in the nucleus. In this study, we unexpectedly found that hc-siRNAs are predominantly present in the cytoplasm. We demonstrated that AG04is loaded with hc-siRNAs in the cytoplasm and the formation of mature AG04/siRNA complexes requires HSP90and the cleavage activity of AG04. Intriguingly, siRNA-binding facilitates the redistribution of AG04into the nucleus, likely through inducing conformational change that leads to the exposure of the nuclear localization signal (NLS). Our findings reveal an unsuspected cytoplasmic step in the RdDM pathway. We propose that selective nuclear import of mature AG04/siRNA complexes is a key regulatory point prior to the effector stage of RdDM In plants, all known small RNAs (sRNAs) are processed by Dicer-like proteins (DCLs). In this study, we explored the possibility that plants produce sRNAs independent of DCLs. We profiled sRNAs in an Arabidopsis dcl quadruple mutant and identified a class of DCL-independent sRNAs (DI-sRNAs). DI-sRNAs have a broader size distribution, mainly ranging from20-60nt. Vast majority of the DI-sRNAs are derived from transposons and other repetitive sequences, most of which are targets of RdDM. DI-sRNAs are associated with AGO4and guide AGO4to homologous sequences to direct DNA methylation. Intriguingly, some DI-sRNAs appear as ladders in size, with identical5’ends but different3’ends, suggesting that DI-sRNAs are processed through3’-5’ trimming of AGO4-bound precursor transcripts. These data indicate that Arabidopsis can produce sRNAs in a DCL-indepenent manner. We propose that DI-sRNAs may serve as the primal signal for the establishment of RdDM.