1. Volume 46, Issue 6, Pages 859-870 (June 2012)
Cytoplasmic Assembly and Selective Nuclear Import of Arabidopsis ARGONAUTE4/siRNA Complexes 
Ruiqiang Ye, Wei Wang, Taichiro Iki, Chang Liu, Yang Wu, Masayuki Ishikawa, Xueping Zhou, Yijun Qi 
Molecular Cell 
Volume 46, Issue 6, Pages (June 2012)
DOI: /j.molcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2012 46, 859-870DOI: (10.1016/j.molcel.2012.04.013)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1 Hc-siRNAs Are Predominantly Present in the Cytoplasm
(A) Quality controls of the preparation of cytoplasmic (C) and nuclear (N) fractions. PEPC, RBP1, HDA19, and histone H3 proteins were detected by western blot, and tRNA and U6 were probed by northern blot. PEPC and tRNA served as protein and RNA markers for the cytoplasmic fraction, and RBP1, HDA19, histone H3 and U6 served as nuclear markers. T, whole-cell extract.
(B) Size distributions of the total, cytoplasmic, and nuclear sRNAs. The abundance of sRNAs was calculated as reads per million nuclear genome-matching sequences (RPMs). Average RPMs from three biological replicates were used to generate the histogram. Error bars indicate SD. nt, nucleotide. See Table S1 for the summary of small RNA datasets.
(C) Scatter plot of the RPM of each miRNA (blue dot) and hc-siRNA (red dot) in the cytoplasmic fraction versus that in the nuclear fraction. A slope = 1 line is shown as reference. Average RPMs from three biological replicates were used to generate the histogram. See Tables S2 and S3 for RPMs of individual miRNAs and hc-siRNAs.
(D) Cytoplasmic and nuclear allocations of representative miRNAs and hc-siRNAs. The numbers shown are RPMs (mean ± SD) of each miRNA and hc-siRNA. See Tables S2 and S3 for complete lists of miRNAs and hc-siRNAs.
(E) Detection of representative miRNAs and hc-siRNAs in total, cytoplasmic, and nuclear extracts by northern blot. Note that 10 or 30 times more extracts were used in the last lane than the first three lanes. The northern blots were stripped and reprobed multiple times. The positions of sRNA size markers, electrophoresed in parallel, are shown to the right of the gels. nt, nucleotide.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

4. Figure 2
Biochemical Characterization of Cytoplasmic and Nuclear Hc-siRNAs
(A) Detection of indicated sRNAs in total, cytoplasmic, and nuclear extracts by 15% native PAGE electrophoresis followed by northern blot. Note that 10 or 30 times more extracts were used for the nuclear fraction. End-labeled synthetic 24 nt double-stranded (ds) and single-stranded (ss) sRNAs, electrophoresed in parallel, were used as size markers.
(B) Fractionation of total, cytoplasmic, and nuclear extracts by size exclusion chromatography. The indicated sRNAs and proteins were detected in each fraction. Fraction numbers are marked above the gel, and size makers are shown to the right. The black arrows indicate fractions where the 480 kDa, 160 kDa, and 67 kDa protein standards were eluted.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

5. Figure 3 AGO4 Loading of Hc-siRNAs in the Cytoplasm
(A) Representative images showing the localization of GFP-AGO4 in Arabidopsis wild-type (Col-0) protoplasts when it is expressed under control of 35S or its native promoter. A construct expressing SV40NLS-mCherry was cotransfected to label the nucleus. Bar = 5 μm. See Figure S2A for more protoplast images.
(B) Northern blot analysis of indicated sRNAs in AGO4 complexes immunoprecipitated from total, cytoplasmic, and nuclear extracts.
(C) A diagram showing the structure of AGO4.
(D) Representative images showing the localization of GFP-AGO4 and GFP-AGO4ΔNLS in the root cells of transgenic Col-0 plants expressing the GFP fusion proteins under the control of AGO4 promoter. Cell walls were stained with propidium iodide (PI) before confocal microscopy, which fluoresces red. Bar = 15 μm.
(E) Northern blot analysis of hc-siRNAs in total extracts and AGO4 immunoprecipitates prepared from Col-0 and indicated transgenic plants. U6 RNA was probed and used as a loading control for total extracts. A silver-stained gel shows that equal amounts of AGO4 complexes were used for northern blot analysis.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

6. Figure 4 Biochemical Mechanism of AGO4 Loading of Hc-siRNAs
(A) Structure of the GP24 siRNA duplex used for in vitro assembly of AGO4 complex.
(B) Synthesis and immunopurification of myc-AGO4 and the mutant proteins. myc-AGO4 and the mutant proteins were synthesized by in vitro translation using BYL. The samples before (input) and after (myc-IP) immunopurification were analyzed by western blot using anti-myc antibody. In parallel, mock-translated BYL was analyzed.
(C) Assembly of AGO4/siRNA complex in cell-free system. myc-AGO4 and the mutant proteins were synthesized by in vitro translation using BYL and incubated with GP24 siRNA duplexes in which the 5′-end nucleotide of either guide (Strand G) or passenger (Strand P) strand was 32P-labeled. Then, immunopurification was performed with anti-myc antibody. RNA samples before (input) and after (myc-IP) immunopurification were analyzed by 15% native PAGE. In parallel, mock-translated BYL was analyzed. The bands labeled with an asterisk likely represent an intermediate product: the guide strand paired with half of the passenger strand after AGO4-mediated cleavage.
(D) Detection of hc-siRNAs (SIMPLEHAT2) in wild-type and mutant AGO4 immunoprecipitates prepared from total (T), cytoplasmic (C), and nuclear (N) extracts by 15% native PAGE electrophoresis followed by northern blot. Silver-stained gels show the amounts of AGO4 complexes that were used for northern blot analysis. End-labeled synthetic 24 nt double-stranded (ds) and single-stranded (ss) small RNAs, electrophoresed in parallel, were used as size markers.
(E) Copurification of HSP90 with AGO4. myc-AGO4 protein was synthesized in BYL and incubated with or without ATPγS and GA. In parallel, mock-translated BYL was incubated in the +ATPγS-GA condition. Immunopurification was performed with anti-myc antibody. Samples before (input) and after (myc-IP) immunopurification were analyzed by immunoblotting using anti-myc or anti-HSP90 antibody.
(F) Copurification of siRNAs with HSP90 in the presence of ATPγS. Immunopurification was performed with anti-HSP90 antibody.
(G) Effect of the addition of GA on the assembly of AGO4/siRNA complexes. Synthesized myc-AGO4 protein was incubated with GP24 siRNA duplex (Strand G was 32P-labeled) in the absence (−) or in the presence (+) of GA. myc-AGO4 proteins were then immunopurified using anti-myc antibody, and copurified RNAs were analyzed by 15% native PAGE (“myc-IP” panel), together with RNAs extracted from the samples before immunopurification (“input” panel). In parallel, mock-translated BYL was analyzed in GA-free condition.
(H) Detection of the expression levels of the four HSP90s by quantitative RT-PCR in Col-0 and pooled RNAi-A or RNAi-C primary transformants. The expression levels of HSP90s were normalized using the signal from the Actin gene. The average (±SD) values from three technical repeats are shown.
(I) Analysis of DNA methylation at AtSN1 and MEA-ISR loci in the indicated HSP90 RNAi lines by bisulfite sequencing. More than 20 clones were sequenced for each sample.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

7. Figure 5 Hc-siRNA Loading Facilitates AGO4 Nuclear Localization
(A) Representative images showing the localization of GFP-AGO4 in Col-0, dcl2-1 dcl3-1 dcl4-2, and ktf1-1 protoplasts. The protoplasts of indicated genotypes were transfected with DNA constructs that express GFP-AGO4 under the control of 35S promoter. See Figure S4 for GFP-AGO4 localization in transgenic plants.
(B) Representative images showing the localization of GFP-AGO4, GFP-AGO4Y390AF391A, and GFP-AGO4D742A in Col-0 protoplasts. A construct expressing SV40NLS-mCherry was cotransfected to label the nucleus. Bars = 5 μm. See Figures S2B and S2C for more protoplast images.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

8. Figure 6 Positional Effect of the NLS on AGO4 Localization
(A) Representative images showing the localization of GFP-AGO4 and GFP-AGO4N-NLS in Col-0 and dcl2-1 dcl3-1 dcl4-2 protoplasts.
(B) Representative images showing the localization of GFP-AGO4Y390AF391A and GFP-AGO4N-NLS/Y370AF371A in Col-0 protoplasts.
(C) Representative images showing the localization of GFP-AGO4SV40-NLS in Col-0 and dcl2-1 dcl3-1 dcl4-2 protoplasts.
A construct expressing SV40NLS-mCherry was cotransfected to label the nucleus. Bars = 5 μm. See Figures S2D–S2F for more protoplast images.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

9. Figure 7
A Revised Model for RdDM Featuring Cytoplasmic Assembly and Selective Nuclear Import of AGO4/siRNA Complexes
In this model, siRNA duplexes are produced in the nucleus by sequential activities of Pol IV, RDR2, DCL3, and other accessory factors. AGO4 might also participate in this process through cleaving Pol IV transcripts to serve RDR2 with better substrates. siRNA duplexes are exported into the cytoplasm. In the cytoplasm, facilitated by HSP90, siRNA duplexes are loaded into AGO4. AGO4-mediated cleavage triggers the removal of the passenger strands, resulting in mature AGO4/siRNA complexes with an accessible NLS. Mature AGO4/siRNA complexes are then imported into the nucleus. In the nucleus, AGO4/siRNA complexes interact with Pol V transcripts and further recruit other factors to mediate DNA methylation.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions