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. 2012 Apr 4;31(7):1704-13.
doi: 10.1038/emboj.2012.20. Epub 2012 Feb 10.

Cytoplasmic Arabidopsis AGO7 accumulates in membrane-associated siRNA bodies and is required for ta-siRNA biogenesis

Virginie Jouannet  1 , Ana Beatriz Moreno, Taline Elmayan, Hervé Vaucheret, Martin D Crespi, Alexis Maizel
Affiliations

Cytoplasmic Arabidopsis AGO7 accumulates in membrane-associated siRNA bodies and is required for ta-siRNA biogenesis

Virginie Jouannet et al. EMBO J. .

Abstract

Formation of trans-acting small interfering RNAs (ta-siRNAs) from the TAS3 precursor is triggered by the AGO7/miR390 complex, which primes TAS3 for conversion into double-stranded RNA by the RNA-dependent RNA polymerase RDR6 and SGS3. These ta-siRNAs control several aspects of plant development. The mechanism routing AGO7-cleaved TAS3 precursor to RDR6/SGS3 and its subcellular organization are unknown. We show that AGO7 accumulates together with SGS3 and RDR6 in cytoplasmic siRNA bodies that are distinct from P-bodies. siRNA bodies colocalize with a membrane-associated viral protein and become positive for stress-granule markers upon stress-induced translational repression, this suggests that siRNA bodies are membrane-associated sites of accumulation of mRNA stalled during translation. AGO7 congregates with miR390 and SGS3 in membranes and its targeting to the nucleus prevents its accumulation in siRNA bodies and ta-siRNA formation. AGO7 is therefore required in the cytoplasm and membranous siRNA bodies for TAS3 processing, revealing a hitherto unknown role for membrane-associated ribonucleoparticles in ta-siRNA biogenesis and AGO action in plants.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
AGO7 accumulates in cytoplasmic foci. (A) Confocal section of a Nicotiana tabacum leaves expressing GFP–AGO7. Cytoplasmic AGO7 foci are indicated by arrowheads. The cytoplasm appears as a thin peripheral layer, while the vacuole (v) fills most of the cell volume. Inset: GFP–AGO7 is not detected in the nucleus (n). (B) Morphology of 3-week-old ago7-1 or p35S:GFP:AGO7/ago7-1 Arabidopsis plants. ago7-1 plants display the typical zippy phenotype (narrow pointed leaves). (C) RNA gel blot analysis of 15 μg of total RNA from 7-day-old wild-type (WT), ago7-1 mutant or four independent p35S:GFP:AGO7/ago7-1 plants. The blot was probed with DNA complementary to ta-siARFs. U6 snRNA served as a loading control. (D) Western blot analysis of 7-day-old p35S:GFP:AGO7/ago7-1 plants. The blot was probed with an anti-GFP. The arrowhead indicates the position of the GFP–AGO7 band (127 kDa). (E) Indirect immunofluorescence detection of GFP–AGO7 in epidermal cells of the root meristem in 7-day-old p35S:GFP:AGO7/ago7-1 plants using an anti-GFP antibody. In meristem cells, the vacuole is not yet formed. The inset represents a non-transgenic plant processed and imaged in the same conditions. (F) Same as in (E) but counterstained with DAPI to mark nuclei (n). (G) Indirect immunofluorescence detection of HA–AGO7 in epidermal cells of the root meristem in 7-day-old pAGO7:HA:AGO7/zip-1 plants using an anti-HA antibody. The lower left inset represents a non-transgenic plant processed and imaged in the same conditions, whereas the upper right one is a higher magnification view. Scale bars: (A, F and G inset) 5 μm; (E, G) 25 μm. Figure source data can be found in Supplementary data.
Figure 2
Figure 2
AGO7 accumulates in membrane-linked cytoplasmic siRNA bodies. (A–F) Confocal sections of Nicotiana tabacum leaves expressing the indicated fluorescent fusion proteins. For each condition, the signals of the individual fluorophores are presented in the first two images whereas the signals are merged in the last image. The arrows indicate the AGO7/siRNA bodies. The dotted squares depict the location of the higher magnification insets. Scale bars: 5 μm.
Figure 3
Figure 3
siRNA bodies colocalize with stress granules after heat shock. (A–D) Confocal sections of Nicotiana tabacum leaves expressing the indicated fluorescent fusion proteins. For each condition, the green and red signals of the same plane are presented in two first images whereas the signals are merged in the last image. The arrows indicate the AGO7/siRNA bodies. The dotted squares depict the location of the higher magnification insets. Images in (C) and (D) were taken tangentially to the cytoplasm surface; n: nucleus. Scale bars: 5 μm.
Figure 4
Figure 4
AGO7 copurifies with a membranous fraction. (A–E) Western blot analysis of 7-day-old pAGO7:HA-AGO7/zip-1 plants. Unless indicated otherwise, the blots were probed with an anti-HA antibody (HA–AGO7: 113 kDa). (A) Analysis of 75 μg of protein from the total, nuclear (N) and post-nuclear (PNS) fractions. (B) Analysis of 75 μg of protein from the low speed supernatant (LSS), soluble fraction (S100) and microsomal fraction (P100). The blot was probed with anti-HA (upper panel) and an antibody against the cytosolic fructose-1,6-bisphosphatase (cFBPase, lower panel). (C) Resolubilization of the microsomal fraction by treatment with detergents (DOC: deoxycholate). In all, 75 μg of protein was analysed. (D) Analysis of 75 μg of protein from the resuspended microsomal fraction treated (+) or not (−) by the proteinase K (Prot. K) before recentrifugation at 100 000 g. (E) Analysis of the different fractions after separation of the resuspended microsomal fraction on a continuous (20–60% w/v) sucrose gradient. The blot was probed with anti-HA (upper panel) and an antibody against calnexin (lower panel). Figure source data can be found in Supplementary data.
Figure 5
Figure 5
miR390 co-fractionates with AGO7. RNA gel blot analysis of 15 μg (A) or 30 μg (B) of RNA from 7-day-old plants. (A) Analysis of miR390 accumulation in the nuclear and post-nuclear fractions of wild-type and zip-1 plants. (B) Analysis of miR390 accumulation in low speed supernatant (LSS), soluble fraction (S100) and microsomal fraction (P100) of wild-type plants. Ethidium bromide (EtBr) staining served as a loading control. Figure source data can be found in Supplementary data.
Figure 6
Figure 6
Nuclear localization impairs AGO7 function in ta-siRNA biogenesis. (A) Schematic representation of AGO7 and the NLS and NES alleles used. (B) Confocal sections of Nicotiana tabacum leaves expressing the indicated fluorescent fusion proteins. On the upper panels are views of the cytoplasm and on the lower panels of the nuclei (n). The arrowheads indicate the AGO7 siRNA bodies. Scale bars: 5 μm. (C) Morphology of 3-week-old syntasi-PDS/zip-1 Arabidopsis plants expressing the indicated construct from the AGO7 promoter. Bleached plants harbour white sectors radiating from the veins. (D) RNA gel blot analysis of 30 μg of total RNA from zip-1 inflorescence expressing the indicated construct from the AGO7 promoter. Each lane represents independent primary transformants (three for each allele). The blot was probed with DNA complementary to ta-siARFs. U6 snRNA served as a loading control and numbers indicate normalized intensities. (E) Morphology of 3-week-old wild-type and zip-1 Arabidopsis plants expressing the indicated construct from the AGO7 promoter. Figure source data can be found in Supplementary data.
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