Arabidopsis Argonaute10 specifically sequesters miR166/165 to regulate shoot apical meristem development - PubMed (original) (raw)
Arabidopsis Argonaute10 specifically sequesters miR166/165 to regulate shoot apical meristem development
Hongliang Zhu et al. Cell. 2011.
Abstract
The shoot apical meristem (SAM) comprises a group of undifferentiated cells that divide to maintain the plant meristem and also give rise to all shoot organs. SAM fate is specified by class III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) transcription factors, which are targets of miR166/165. In Arabidopsis, AGO10 is a critical regulator of SAM maintenance, and here we demonstrate that AGO10 specifically interacts with miR166/165. The association is determined by a distinct structure of the miR166/165 duplex. Deficient loading of miR166 into AGO10 results in a defective SAM. Notably, the miRNA-binding ability of AGO10, but not its catalytic activity, is required for SAM development, and AGO10 has a higher binding affinity for miR166 than does AGO1, a principal contributor to miRNA-mediated silencing. We propose that AGO10 functions as a decoy for miR166/165 to maintain the SAM, preventing their incorporation into AGO1 complexes and the subsequent repression of HD-ZIP III gene expression.
Copyright © 2011 Elsevier Inc. All rights reserved.
Figures
Figure 1
AGO10 predominantly recruits miR166/165. (A) Two-step affinity purification of epitope-tagged AGO10-containing RISCs. (B) Cloning and sequencing of AGO10-associated sRNAs. The sRNAs recovered from AGO10 complexes were spiked with 32P-labeled internal 21 and 24 nt sRNA controls and traced throughout the entire cloning process. (C) Approximately 90% of AGO10-bound miRNAs were miR166/165. (D–F) The specific AGO10-miR166/165 interaction was confirmed in Arabidopsis (D) and in N. bentha (E and F). sRNA blots were conducted with total RNA and sRNAs recovered from immunoprecipitated AGO complexes (IP). Western blot analyses were done with the crude extract and aliquots of the IP products using anti_-_YFP or -Myc antibodies. A cross-reacting band (**) served as a loading control. See also Fig. S1.
Figure 2
Few mutations in the miR166 sequence compromised miRNA loading into AGO10. (A) Schematics of point mutations in miR166 and its * strand. Predicted foldback of miR166a (Left panel). Paired single mutations in miR166 (red) and its * strand (blue) (Middle panel). The outside region of the miR166/166* duplex is shown in black. Numbers (i.e. 1–21) were given next to miR166/166* to show its orientation. −1 and −2 indicate their relative positions to the start of the miR166* strand. (B–D) Loading of miR166 mutants into AGO10 in N. bentha. Analyses of sRNA and western blots were conducted as in Figure 1E. (E) The relative mean signals of miR166 mutants/AGO10 were normalized to that of miR166/AGO10 with ±SD from seven experiments. Note: miR166 C11G *G9C was not detected in the input. See also Fig. S2.
Figure 3
The internal structure of miR166/166* determines the specific AGO10-miR166 association. (A) Predicted foldbacks of pre-miR390b and -miR168a and chimeric precursors expressing miR166. miR166 (red) and its * strand (blue) are shown. The mutated nucleotides in the miR168*- and miR390*-like strands are shown in green. (B) Primer extension experiments were conducted with total RNAs prepared from N. bentha transfected with the indicated constructs. (C and D) Change of the miR166/166* structure dramatically decreased the loading of miR166 to AGO10 (C), but not to AGO1(D). Analyses of sRNA and western blots were conducted as in Figure 1E. The relative mean ratio of miR166/AGO10 (or AGO1) was normalized to that obtained with pre-miR166a with ±SD from five repeats (bottom panels). See also Fig. S3.
Figure 4
Deficient loading of miR166 into AGO10 causes pinhead phenotypes in the Col-0 background. (A) Shared morphological phenotypes of 35S-miR166a, -miR166/166*390 and -miR166/390*-like390 plants. Photographs were taken of 10-day-old seedlings. Two representative lines are shown for each construct. (B) miR166 level was measured by sRNA blot analysis. (C) Transcript levels of selected AGO and HD-ZIP family genes were measured by northern blot analysis. (D) Unique pinhead phenotypes of 35S-miR166/390*-like390 plants. (E and F) Deficient loading of miR166 from the miR166/390*-like390 precursor into AGO10 (E) but not AGO1 (F) in Arabidopsis. Analyses of sRNA blot and western blot (using an anti-Flag antibody) were conducted as in Figure 1D. The exposure times for AGO10 and AGO1 protein blots were 30 and 5 seconds, respectively. The relative mean ratio of miR166/AGO10 (or AGO1) was measured as in Figure 3 with _±_SD from three experiments (bottom panels). (G ) Correlation of the imbalanced loading of miR166/165 into AGO1/AGO10 with pinhead phenotype. See also Fig. S4.
Figure 5
Sequestration of miR166/165 from expression domains of AGO10, but not AGO1, by target mimicry rescues the ago10pnh-2 phenotype. (A) AGO10 Q885* encoded by ago10pnh-2 did not bind to miR166 in N. bentha due to improper protein folding. Analyses of sRNA and western blots were conducted as in Figure 1E. (B and C) ago10 mutation resulted in a significant increase in miR166 binding by AGO1 in Arabidopsis. sRNA blot analyses were conducted with total RNA (input) and sRNA recovered from the AGO1 complexes (IP). Western blot assays were performed using an anti-AGO1 antibody. A cross-reacting band (**) served as a loading control. The relative signal ratio of miR166 to miR159 in AGO1 complexes was normalized to that obtained from the wild type Ler or the PAGO10-HF-AGO10 complemented lines with ±SD from three experiments (bottom panels). (D) Defective SAM was rescued in ago10pnh-2 plants by miR166/165 target mimicry expressed from the promoters of AGO10, but not AGO1. The ratios of defective SAM are shown as mean ±SD from three replicates (n > 200/each replicate). (E and F) Levels of miR166/165 and their target transcripts were measured by RNA blot assays (E) and real-time RT-PCR (F). The relative level of HD-ZIP transcripts was normalized to that in Ler plants with ±SD from four experiments. See also Fig. S5.
Figure 6
AGO10 rescues the ago10pnh-2 mutant by sequestering miR166/165 from AGO1. (A) AGO10 DDH mutants maintained miR166/165-binding capacity. Assays of sRNA blot and western blot (using anti-Flag antibody) were conducted as in Figure 1D. (B) RISC reconstitution assays of AGO10 and AGO10 DDH mutants. AGO1 was included as a positive control. (C) Non-catalytic AGO10 rescued the ago10pnh-2 mutant as efficiently as catalytic AGO10. The pinhead ratios are shown as mean ±SD from 16 lines (n > 200/line). (D and E) Levels of miR166/165, AGO10 and HD-ZIP III transcripts were measured by analyses of sRNA and northern blots (D) and real-time RT-PCR (E). The relative level of HD-ZIP transcripts was normalized as in Figure 5F.(F) AGO10 sequestered miR166 from AGO1. Analyses of sRNA blot and western blot (using the anti-AGO1 or anti-Flag antibody) were conducted as in Figure 1D. (G) The relative binding of miR166 by dual-tagged AGO10 was normalized to that of miR166/AGO1 isolated from ago10 pnh-2; PAGO10-HF-AGO10 plants with ±SD from three experiments. See also Figs. S5 and 6.
Figure 7
AGO10 maintains the SAM by specifically decoying miR166/165 to upregulate HD-ZIP family genes. SAM (red crescent) is specified by the HD-ZIP transcription factors (brown rectangle) located within the AGO10 expression niche. AGO10 expression (light green) is limited to the provasculature and the adaxial side of the cotyledons. AGO1 is expressed ubiquitously in the whole embryo (grey), partially overlapping the AGO10 expression domain. AGO10 is a positive regulator of HD-ZIP family genes (red arrow), whereas AGO1 is a negative regulator (white T). The terminated SAM is indicated by a red stop sign. miR166/165 and MIM166/165 are shown as yellow and white bars.
Comment in
- Argonaute10 as a miRNA locker.
Manavella PA, Weigel D, Wu L. Manavella PA, et al. Cell. 2011 Apr 15;145(2):173-4. doi: 10.1016/j.cell.2011.03.045. Cell. 2011. PMID: 21496638
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