The Arabidopsis RNA-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 5' 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 5' 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 AGO-associated proteins in influencing epigenetic modifications.

Figure 1.

The AGO4 Group Proteins Bind 24-Nucleotide sRNAs Produced by the RdDM Pathway. (A) Immunoblots demonstrate that antipeptide antibodies to AGO4, AGO6, and AGO9 are specific, and the proteins show decrease in some RdDM mutants. (B) RNA stability is not affected in the RNA interference mutants in which protein levels are decreased. The vertical axis represents the ratio of the average C(t) value of each AGO mRNA to ACTIN2 levels. Error bars represent an se of the ratio. (C) AGO4, AGO6, and AGO9 preferentially bind 24-nucleotide sRNAs. Each panel contains the sRNA size profile of the total population or IP data sets. FLAG AGO4 (454) depicts the FLAG AGO4 C data set. (D) The AGO4 group proteins have a 5′ nucleotide bias toward adenosine. Each panel denotes the percentage of sRNAs with 5′ nucleotide identities. For the total population, AGO4 IP and AGO9 IP immunopurified with the antipeptide antibody, and the 5′ nucleotide composition for the 24-nucleotide size class is indicated below the appropriate bar. Rep, replicate.

Figure 2.

The AGO4 Group Preferentially Associates with Repeat and Heterochromatin sRNA Loci. The vertical axis indicates the number of sRNA loci with >50% of the locus overlapping the genomic feature indicated above the chart. Error bars represent 1 sd above and below the average number of loci that overlap a random rearrangement of the genomic features on the genome (based on 100 randomizations).

Figure 3.

The AGO4 Group Proteins Accumulate in a Tissue-Specific Fashion. (A) Immunoblots indicate that the AGO4 group proteins accumulate in both floral and silique tissue. (B) Only PAGO4:GUS was detectable in leaf tissue; Col-0 lacks the GUS transgene. (C) PAGO4:GUS, PAGO6:GUS, and PAGO9:GUS show differential expression patterns within embryos. (D) PAGO4:GUS and PAGO9:GUS show differential expression patterns within mixed-stage floral tissue and siliques. (a) to (e) and (g) to (j) are floral tissue; (f) and (k) are siliques. Black bars = 500 μm; red bars = 250 μm. (E) Immunohistochemistry on transverse sections of mixed-stage floral tissue shows specific AGO4 protein expression in developing ovules and vascular tissue. Only background staining was observed with the AGO4 antipeptide antibody on ago4-3 floral tissue. v, vascular tissue; o, ovules; p, petals. Bar = 100 μm.

Figure 4.

Functional Divergence among AGO4, AGO6, and AGO9. (A) Immunoblot analysis (αμFLAG antibody) shows a similar level of AGO4, AGO6, and AGO9 when expressed under the AGO4 promoter. AGO1 levels were used as a loading control. (B) RNA gel blot analyses of PAGO4:FLAG AGO6 and PAGO4:FLAG AGO9 in the ago4-3 background (bkgd) show locus-specific complementation of AGO4-dependent sRNAs. (C) Bisulfite analyses of AtSN1 and SIMPLEHAT2 indicate that asymmetric methylation complementation by AGO6 and AGO9 correlates with the observed sRNA accumulation at each locus (PAGO4:FLAG AGO9 partially complements, while PAGO4:FLAG AGO6 does not complement). For AtREP2, PAGO4:FLAG AGO9 fully complemented CpHpG and CpHpH methylation, while PAGO4:FLAG AGO6 had partial, but significant, complementation of CpHpH but not CpHpG. The different cytosine contexts are indicated above the top panel. The vertical axis represents the average percentage of methylated cytosines (per context). Error bars represent 95% confidence limits. The SIMPLEHAT2 locus contained only cytosines in a CpHpH context. Sample labels appear below the AtREP2 panel. H represents the nucleotides A, T, or C.

Figure 5.

The AGO4 Group Proteins Are Capable of Interacting with the Largest Subunit of POLV. Each AGO was immunopurified using the FLAG epitope, and equal amounts of AGO protein were present on the agarose beads (αFLAG beads panel). The αPOLV input panel shows an equal input of the POLV largest subunit, except in the nrpe1/drd3-1 mutant. The αPOLV co-IP panel contains the FLAG-AGO recombinant protein and any interacting proteins. This was subjected to immunoblotting using the POLV antibody. PAGO4:FLAG AGO6 repeatedly showed the greatest degree of co-IP interaction.

Figure 6.

Locus-Specific sRNA Accumulation by AGO6/AGO9 Expression Does Not Correlate with the AGO4^D660A Mutant. AGO4-dependent loci were tested by RNA gel blot analyses and are denoted by locus numbers. All loci required NRPE1/DRD3 for sRNA accumulation. Most require an AGO4 catalytic PIWI domain. Genomic locations for each locus are listed in Supplemental Table 2 online.