A Suppressor Screen for AGO1 Degradation by the Viral F-Box P0 Protein Uncovers a Role for AGO DUF1785 in sRNA Duplex Unwinding

Abstract

In Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing and is a key component in antiviral responses. The polerovirus F-box P0 protein triggers AGO1 degradation as a viral counterdefense. Here, we identified a motif in AGO1 that is required for its interaction with the S phase kinase-associated protein1-cullin 1-F-box protein (SCF) P0 (SCF^P0) complex and subsequent degradation. The AGO1 P0 degron is conserved and confers P0-mediated degradation to other AGO proteins. Interestingly, the degron motif is localized in the DUF1785 domain of AGO1, in which a single point mutation (ago1-57, obtained by forward genetic screening) compromises recognition by SCF^P0 Recapitulating formation of the RNA-induced silencing complex in a cell-free system revealed that this mutation impairs RNA unwinding, leading to stalled forms of AGO1 still bound to double-stranded RNAs. In vivo, the DUF1785 is required for unwinding perfectly matched siRNA duplexes, but is mostly dispensable for unwinding imperfectly matched miRNA duplexes. Consequently, its mutation nearly abolishes phased siRNA production and sense transgene posttranscriptional gene silencing. Overall, our work sheds new light on the mode of AGO1 recognition by P0 and the in vivo function of DUF1785 in RNA silencing.

Figure 1.

AGO1 in the sup149.1 Mutant Is Insensitive to P0-Mediated Degradation. (A) Top panel: Immunoblot analysis of AGO1 content in mock (−) or P0 induced (+) 7-d-old seedlings, grown on horizontal solid medium, with or without β-estradiol. Probing with the ACTIN antibody and Coomassie blue (CB) staining were used as loading controls. Middle panel: P0 expression is measured by RNA gel blot; loading control is shown by staining the membrane with methylene blue (MB). Bottom panel: AGO1 mRNA level in the same samples measured by RT-qPCR. Levels are displayed relative to Col-0. “@” indicates hybridization with antibody or DNA probe. (B) Top panel: Root length measurement of vertically grown seedlings, after 9 d on either mock (−) or β-estradiol containing (+) medium. ANOVA was performed to compare genotypes and treatment, P < 0.001. Bottom panel: Representative individual seedlings in mock or P0 induced condition, after 7 d. Right panel: Representative individual seedlings in mock or P0 induced conditions, after 9 d. (C) AGO1 and SDE3 protein content in mock (−) or P0 induced (+) 7-d-old seedlings. Coomassie blue staining was used as a loading control. “@” indicates hybridization with antibody. (D) Cartoon depiction of the sup149.1 mutation, a G-to-A transition in position 1112, in the fourth exon of the AGO1 coding sequence. This point mutation leads to the replacement of a glycine (GGC) by an aspartic acid (GAC) in position 371 of the AGO1 protein. See Supplemental File 1 for uncropped source images for immunoblots.

Figure 2.

The Conserved DUF1785 of AGO1 Contains the P0 Degron. (A) Schematic representation of the AGO1 deletion constructs used to assay sensitivity to P0-myc degradation in N. benthamiana. Each construct contains a GFP at the C terminus and is under the control of the CaMV 35S promoter. The name of each AGO1 protein domain is indicated on top. PAZ, Piwi-Argonaute-Zwille; L2, Linker 2; MID, middle domain. (B) Each GFP-AGO1 deletion construct was transiently expressed with (+) or without (−) coinfiltration of the 35S:P0-myc construct. Fusion protein levels were assessed by immunoblot using cMyc and GFP antibodies. The @GFP split panel corresponds to the same membrane but with two different exposures. “@” indicates hybridization with antibody. (C) MUSCLE alignment of Arabidopsis AGO proteins in the DUF1785 region. Amino acid conservation is displayed in blue, the most intense being the most conserved. The 18 amino acids that constitute the P0 degron in AGO1 were defined by boundaries of the degradable versus nondegradable deletion constructs (boxed in red). AGOs identities and deletion constructs depicted by arrows are indicated. Protein domains are highlighted in orange (DUF1785) and yellow (PAZ). (D) Alanine scanning through the 18 amino acids of the P0 degron. Each residue was mutated to an alanine, in the context of the 35S:ΔNΔC-GFP construct. Each mutant was transiently expressed in N. benthamiana with (+) or without (−) coinfiltration of the 35S:P0-myc construct. ΔNΔC-GFP level was assessed by immunoblot using a GFP antibody (@). Asterisks indicate mutations abolishing P0-mediated degradation. (E) Mutation of the conserved glycine (identified in ago1-57) residue in AGO2 is sufficient to block its degradation by P0-myc. The ago1-57 equivalent (G371D) was introduced in a pAGO2:Venus-AGO2 genomic construct to obtain the G352D construct. Either the wild-type or the mutated Venus-AGO2g construct was transiently expressed with (+) or without (−) coinfiltration of the 35S:P0-myc construct. Levels of the Venus-AGO2 protein were monitored by immunoblot using an AGO2 antibody (@). See Supplemental File 1 for uncropped source images for immunoblots.

Figure 3.

The SCF^P0 Only Poorly Associates with the ago1-57 Mutant Protein. (A) Immunoprecipitations of P0-myc were performed on 13-d-old seedlings, cultivated in liquid medium MS + 1% sucrose with either DMSO, 20 μM β-estradiol, or 20 μM β-estradiol + 20 μM MLN4924 for 8.5 h. P0-myc refers to the line containing wild-type AGO1, while P0-myc ago1-57 contains the point mutant version obtained from the cross. P0-myc LP1 contains two alanines in position 63/64 instead of the minimal F-box motif LP, thereby precluding its association to the SCF. IP experiments demonstrate association of P0-myc with wild-type AGO1, when the neddylation inhibitor MLN4924 is added to the culture. Association is lost with the ago1-57 mutant protein, while the LP1 mutant is neither able to associate with the SCF nor with wild-type AGO1. “@” indicates use of a specific antibody for hybridization or immunoprecipitation. (B) Immunoprecipitations of P0-myc were performed on 10-d-old seedlings as described in (A) for 24 h. Separation was performed on a 4 to 12% acrylamide gradient gel for AGO1, CUL1, and ASK1 blotting (Coomassie blue stain CB1) and on a 15% acrylamide gel for RBX1 and cMyc blotting (Coomassie blue stain CB2). “@” indicates use of a specific antibody for hybridization or immunoprecipitation. (C) Predictive structural model of Arabidopsis AGO1 based on known structure of eukaryotic AGOs. Domains are color-coded as in Figure 2A. G8 (G371) and L11-N12 of the degron are shown in red in the context of AGO1 structure, and the DUF1785 fold is shown in orange. See Supplemental File 1 for uncropped source images for immunoblots.

Figure 4.

Phenotypic and Molecular Characterization of the ago1-57 Mutant. (A) Top panel: Representative mutant plants, grown on soil for 32 d. Bottom panel: Schematic AGO1 sequence represents names, positions, and mutated amino acids of selected mutants; ago1-57 is represented in red. (B) Leaf series of Col-0 and ago1-57 21-d-old plants. Leaves are arranged from left to right in order of emergence. (C) RT-qPCR analysis of representative AGO1-miRNA target mRNA in Col-0 (gray), ago1-57 (orange), and ago1-27 (green). Total RNA samples were extracted from 5-week-old rosettes grown on soil and RT-qPCR was conducted on four individual biological replicates for each genotype. Expression levels are shown relative to Col-0 for the two mutants. For each target, the relevant miRNA is indicated in brackets. Compared to ago1-27, ago1-57 displays very little change in target mRNA levels. A t test was performed to compare both mutant genotypes to Col-0. Significant differences in RNA abundance are displayed above each pairwise combination. ***P < 0.001 and **P < 0.01. (D) RNA gel blot analysis of the steady state accumulation of corresponding miRNAs from (C) and siRNA255 originating from the TAS1 locus. Rosette and flower tissues were used for this analysis and simultaneously blotted on three separate membranes probed independently, each stripped and reprobed several times. For the RNA gel blot analysis conducted on rosettes, equal amounts of RNA from the four biological replicates were mixed and 40 μg was loaded on a gel for each genotype. Each miRNA is indicated on the right side of the image. P, passenger strand; G, guide strand. While guide strand accumulation remains mostly unaffected, ago1-57 typically shows overaccumulation of most passenger strand miRNA. “@” indicates hybridization with the indicated DNA probe.

Figure 5.

The ago1-57 Mutation Induces Stacking of sRNA Duplexes inside AGO1 during RISC Formation. (A) Small RNA duplex sequences used in (B) and (C). Sequence of the mature miRNA is shown in red in 5′→3′ orientation, while the paired passenger strand is depicted in blue. Paired nucleotides are joined by a bar and wobble paired nucleotides by a dot. In these assays, the guide strand (red) is ³²P labeled at the 5′ extremity and annealed to the cold passenger (blue) strand. (B) Effect of the ago1-57 point mutation on the generation of single-stranded (ss) guide sRNAs, in the cell-free RISC formation system using evacuolated BY2 extracts (BYL). Either wild-type NtAGO1 or the ago1-57-equivalent mutant was expressed in BYL by in vitro translation and incubated for 30 min with indicated sRNA duplexes containing ³²P-labeled guide strands. Resultant RNAs were extracted and analyzed on native acrylamide gel, allowing differentiation between residual substrate double-stranded (ds) and processed single-stranded sRNA. Mock refers to a BYL extract with labeled sRNA but without NtAGO1. This assay was repeated for several miRNA duplexes as well as two identical siRNA perfect match duplexes, differing only in size by one added nucleotide. The ago1-57 mutation impairs accumulation of several single stranded miRNA, but also of the tested siRNAs, regardless of their size (C) Effect of the ago1-57 point mutation in the removal of the passenger strand from the sRNA duplexes. Experiment was performed as described in (B), and Flag-tagged NtAGO1 was further purified with @Flag antibodies. Total (input) or AGO1-bound RNA (@Flag-IP) was analyzed by native acrylamide gel electrophoresis. The ago1-57 mutation induces retention of duplexed sRNA inside AGO1 to varying degree, thereby decreasing the generation of single-stranded sRNAs loaded into RISC.

Figure 6.

In Vivo Analysis of sRNA Accumulation in the ago1-57 Mutant. (A) Differential analysis of Col-0 normalized sRNA reads (JBT17-18 and JBT23-24) compared with ago1-57 normalized sRNA reads (JBT19-20 and JBT25-26). Left panel: Normalized total RNA libraries. Right panel: Normalized @AGO1 IP libraries. Abundance (mean of normalized counts) is displayed on the horizontal axis and log₂ fold change on the vertical axis. Loci with an adjusted P value lower than 0.05 are highlighted in red. (B) Venn diagram depicting overlap between sRNA generating loci that are either enriched or depleted in ago1-57 compared with the wild type, in total and @AGO1 IPs. See Supplemental Data Set 1 for corresponding loci and sRNA count. (C) Box plot representation of accumulation of sRNA reads over miRNA 5p and 3p annotations. The vertical axis represents the absolute value of the log_e 5p/3p ratio multiplied by −1, while library identities are indicated below the box plot. Only miRNAs with at least 100 reads overlapping the 5p or the 3p in at least one library were considered (n = 63 out of the 93 miRNA precursors with both 5p and 3p annotation in miRBase V21). In order to avoid division by 0, read count values were transformed into pseudo-counts by adding 1 to all values. The closer the box plot is to 0 the more miRNA 5p and 3p annotations have similar amounts of overlapping reads. (D) In vivo analysis of accumulation of diverse sRNAs in Col-0 and three ago1 point mutants, in total RNA and AGO1 immunoprecipitates. ago1-38 and ago1-27 were used, as they presented moderate growth defects and similar protein accumulation to Col-0 and ago1-57, and were therefore comparable. Top panel: @AGO1 IP was performed from 18-d-old seedlings to document accumulation of AGO1-bound sRNA. Both total RNA and RNA recovered in the IP were extracted. Both ago1-57 and ago1-38 appear to accumulate passenger small RNA in the total RNA fraction, for several of the considered loci. On the other hand, ago1-57 retains star strand inside the in vivo AGO1 RISC complex for all considered loci. The ago1-57 mutant also displays products below 21 nucleotides. Bottom panel: AGO1 protein level from both total extracted and IP buffer-extracted proteins, shown as an estimate of the amount of AGO1 that could be immunoprecipitated. “@” indicates hybridization with the indicated DNA probe, or use of a specific antibody for immunoprecipitation. See Supplemental File 1 for uncropped source images for immunoblots. (E) Native In vivo analysis of RNA species found in the AGO1 IP (@) separated on a native acrylamide gel. Top panel: RNA was recovered from AGO1 IPs performed on 11-d-old seedlings, and ³²P labeled at the 5′ extremity. Resultant RNAs were analyzed on native acrylamide gel, allowing differentiation between double-stranded (ds) and processed single-stranded (ss) sRNA. Synthetic siR255/siR255* was used as a size control, either for double-stranded species (annealed and nondenatured) or single-stranded species (heat-treated before loading). The ago1-57 protein contains more global double-stranded species and less single-stranded species then the wild-type AGO1. Bottom panel: AGO1 protein level from IP buffer-extracted proteins, shown as an estimate of the amount of AGO1 that could be immunoprecipitated. “@” indicates hybridization with the AGO1 specific antibody or use of the same antibody for immunoprecipitation.

Figure 7.

The ago1-57 Mutation Impairs Secondary sRNA Production, Except for tasiRNAs. (A) Mapping of sRNA reads on TAS2 and three PPR genes (At1g63080, At1g63130, and At1g62930) targeted by the TAS2-derived tasiRNA: tasiR2140 [also named TAS2-3′D6(−)]. Positions of known small RNA triggers are indicated by dashed lines (B) Phasing of sRNAs over the TAS2 transcript in Col-0 and ago1-57. (C) Abundance of tasiR2140 guide strand and passenger strand in Col-0 seedlings compared with ago1-57, in total RNA and @AGO1 IP. (D) Comparison of the log₁₀ guide/passenger ratio in Col-0 and ago1-57, calculated for both total RNA and AGO1-IP RNA samples. (E) Comparison of TAS2, At1g63080, At1g63130, and At1g62930 mRNA accumulation in Col-0, ago1-57, and ago1-27 seedlings. Total RNA was extracted from 2-week-old seedlings. A t test was performed to compare mutants to Col-0. Significant differences in RNA abundance are displayed above each pairwise combination. ***P < 0.001 and ** P < 0.01. (F) Comparison of the GUS activity measured in L1, L1/ago1-57, and L1/ago1-27 lines.