TheArabidopsisRNA-Directed DNA Methylation Argonautes Functionally Diverge Based on Their Expression and Interaction with Target Loci  

Abstract

Argonaute (AGO) effectors of RNA silencing bind small RNA (sRNA) molecules and mediate mRNA cleavage, translational repression, or epigenetic DNA modification. In many organisms, these targeting mechanisms are devolved to different products of AGO multigene families. To investigate the basis of AGO functional diversification, we characterized three closely related Arabidopsis thaliana AGOs (AGO4, AGO6, and AGO9) implicated in RNA-directed DNA methylation. All three AGOs bound 59 adenosine 24-nucleotide sRNAs, but each exhibited different preferences for sRNAs from different heterochromatin-associated loci. This difference was reduced when AGO6 and AGO9 were expressed from the AGO4 promoter, indicating that the functional diversification was partially due to differential expression of the corresponding genes. However, the AGO4-directed pattern of sRNA accumulation and DNA methylation was not fully recapitulated with AGO6 or AGO9 expressed from the AGO4 promoter. Here, we show that sRNA length and 59 nucleotide do not account for the observed functional diversification of these AGOs. Instead, the selectivity of sRNA binding is determined by the coincident expression of the AGO and sRNA-generating loci, and epigenetic modification is influenced by interactions between the AGO protein and the different target loci. These findings highlight the importance of tissue specificity and AGOassociated proteins in influencing epigenetic modifications.

Dicer-independent RNA-directed DNA methylation in Arabidopsis

“…Among them, AGO4/6/9 were shown to be involved in epigenetic silencing. Although AGO9 mainly functions in reproductive tissues [33], AGO4 and AGO6 are more universally expressed and independently affect DNA methylation at RdDM loci [34]. A recent study has shed light on the molecular mechanism involved in pairing between the AGO-siRNA complex and the scaffold RNA generated by Pol V, and the subsequent recruitment of DNA methyltransferases [35].…”

Roles of DICER-LIKE and ARGONAUTE Proteins in TAS-Derived Small Interfering RNA-Triggered DNA Methylation

“…Small RNA datasets used in this study are as described previously Mi et al, 2008;Mosher et al, 2008;Havecker et al, 2010).…”

“…The majority of hc-siRNAs are 24 nucleotides in length and are derived from transposons and repetitive elements. After being generated by the cooperative actions of plant-specific DNA-DIRECTED RNA POLYMERASE IV (PolIV), RNA-DEPENDENT RNA POLYMERASE2 (RDR2), and DCL3, hc-siRNAs are loaded into AGO4 and AGO6 complexes to trigger RNA-directed DNA methylation (RdDM; Qi et al, 2006;Zheng et al, 2007;Havecker et al, 2010;Law and Jacobsen, 2010). In the RdDM pathway, both PolV-dependent nascent scaffold transcripts and hc-siRNAs are essential to guide de novo cytosine DNA methylation, which maintains the silencing of repeats (Wierzbicki et al, 2008;Wierzbicki et al, 2009).…”

Repeat elements and the Arabidopsis DNA methylation landscape

“…For example, the nature of the primary transcripts required to initiate RNAi remain ill defined, and the mechanism by which DRM2 is recruited to target loci is unknown. Furthermore, as shown for AGO4, AGO6 and AGO9, members of a given protein family may participate to different degrees in RdDM as a result of functional diversification (Zheng et al, 2007;Havecker et al, 2010). Moreover, the observation that Pol IV and Pol V are not involved in the production of strand-specific clusters of 24-nt siRNAs that match long inverted duplications (Kasschau et al, 2007;Zhang et al, 2007) implies that at least in this case, siRNAs must derive from transcripts produced by one of the three canonical RNA polymerases, presumably Pol II.…”