Volume 11, Issue 8, Pages 1008-1023 (August 2018) Structural Flexibility Enables Alternative Maturation, ARGONAUTE Sorting and Activities of miR168, a Global Gene Silencing Regulator in Plants  Taichiro Iki, Antoine Cléry, Nicolas G. Bologna, Alexis Sarazin, Christopher A. Brosnan, Nathan Pumplin, Frédéric H.T. Allain, Olivier Voinnet  Molecular Plant  Volume 11, Issue 8, Pages 1008-1023 (August 2018) DOI: 10.1016/j.molp.2018.05.006 Copyright © 2018 The Author Terms and Conditions

Figure 1 Flexible Base-Pairing Configurations between miR168a and miR168a*. (A) miR168 5’/3’ isomer accumulation in various Arabidopsis tissues, extracted from publicly deposited sRNA-seq data for floral tissues, leaves, and seedlings; values are absolute read counts. (B and C) Secondary structure of Arabidopsis pri-miR168a. The dsRNA motif enabling possible alternative base-pairing patterns is outlined by a square (B) and nucleotide substitutions (m1-4) introduced within miR168a* to stabilize metastable state I, II, or III are shown (C). (D–G) NMR measurement of miR168a duplexes. (D) Predicted secondary structures of synthetic WT or m4 miR168a duplexes analyzed by NMR. Section of a 2D (1H-1H) NOESY spectrum recorded with the miR168a WT (E) or m4 (F) duplex at 283°K with 25 mM MgCl2, or with the WT miR168a duplex at 303°K without MgCl2 (G). Imino proton resonances from (G.U) 1 and (G.U) 2 base pairs are shown in green and red, respectively. Figure 1E shows a reference experiment used to visualize imino proton resonances of both GU1 and GU2 base pairs. Figure 1F shows a control experiment to distinguish imino proton resonances coming from GU1 and GU2. As GU2 was abolished by the m4 mutation, only GU1 resonances are visible. Figure 1G shows a broadening of GU2 imino cross-peaks suggesting that this part of the stem is dynamic. Molecular Plant 2018 11, 1008-1023DOI: (10.1016/j.molp.2018.05.006) Copyright © 2018 The Author Terms and Conditions

Figure 2 Effects of Flexibility-Triggered Alternative Base-Pairing Configurations on miR168a Isoform Accumulation. (A) miR168a and miR168a* isoform accumulation upon WT or m1-4 pri-miR168a expression in miR168a-2 plants. Northern analysis of RNA extracted from rosette leaves of representative T2 plants. miR171 and U6 serve as loading controls. (B) AGO1 protein levels in leaves analyzed in (A). Coomassie brilliant blue (CBB) staining serves as a loading control. (C) Proposed structural alterations caused by m1-4 and their predicted impact on miR168a processing. Red arrows indicate thermodynamically favorable structures. Molecular Plant 2018 11, 1008-1023DOI: (10.1016/j.molp.2018.05.006) Copyright © 2018 The Author Terms and Conditions

Figure 3 Mapping of Dicer Cleavage Positions on miR168a Precursors Stabilized into Alternative Base-Pairing Configurations. (A) Processing of Arabidopsis miR168a. The primary cleavage near the base releases pre-miR168a, and the following one, near the apical loop, releases mature miR168a duplexes together with the apical loop byproduct. (B) Identification of boundary nucleotides exposed by Dicer cleavages via intramolecular RNA ligation. The miR168a fragments were circularized with T4 RNA ligase (1), cDNA was synthesized with a primer on the apical loop region (2), and DNA fragments were amplified by PCR, cloned, and sequenced (3). (C) miR168a duplex length measurement using nucleotides exposed on the apical loop borders. Boundary sequences of miR168a/miR168a* are shown in blue and red, respectively. (D) Duplex lengths from WT or m1-4 pri-miR168a measured via apical loop analysis. The values below each diagram indicate the number of independently sequenced clones. Molecular Plant 2018 11, 1008-1023DOI: (10.1016/j.molp.2018.05.006) Copyright © 2018 The Author Terms and Conditions

Figure 4 Effects of Introduced miR168a Flexible Motif on pre-miR319 Processing and AtAGO1-Loading. (A) The pri-miR319/168 chimeric construct containing the WT or m1-4 miR168a motif. (B) pri-miR319/168 hybrids were transiently expressed in N. benthamiana leaves with (+) or without (-) co-expressed FLAG-HA-tagged Arabidopsis AGO1. RNA and proteins were extracted from leaves (input) or from anti-FLAG immuno-precipitates (FLAG-IP), and analyzed via RNA or protein blot. miR165/U6 and CBB serve as loading controls, for RNA and protein blots, respectively. Molecular Plant 2018 11, 1008-1023DOI: (10.1016/j.molp.2018.05.006) Copyright © 2018 The Author Terms and Conditions

Figure 5 Duplex Structural Alterations and Associated Length Variations Regulate miR168a Transitivity Potential and Selective Sorting into AGO1, AGO2, or AGO10. (A) miR168a transitivity potential in rosette leaves of WT or miR168a-2 plants expressing WT or m1-4 pri-miR168a, measured by northern analysis using a mixture of three DNA oligonucleotide probes complementary to abundant AGO1-derived secondary siRNAs. U6 serves as a loading control. (B) Arabidopsis AGO1 or AGO2 were immuno-purified from seedlings of T2 miR168a-2 plants expressing WT or m1-4 pri-miR168a, using anti-AGO1 or -AGO2 antibody. U6 and CBB staining serve as RNA and protein loading controls, respectively. (C) AGO1, AGO2, or AGO10 produced in BYL by in vitro translation were incubated with synthetic duplexes containing 21∼22-nt 32P-labeled miR168a paired with 21∼22-nt miR168a*. AGO-loaded, single-stranded (ss) miR168a migrates faster than the duplex substrate (ds) in native gels. (D) In vitro loading of 22/21-nt/nt WT or m1-4 miR168a duplexes into AGO10. (E) Isomer-specific analysis of AGO-bound miR168 using publicly available AGO-IP sRNA-seq data, expressed as percentages. The averages of multiple sRNA-seq values for each AGO protein are shown. See Supplemental Figure 4A for individual sRNA-seq values. Molecular Plant 2018 11, 1008-1023DOI: (10.1016/j.molp.2018.05.006) Copyright © 2018 The Author Terms and Conditions

Figure 6 22-nt miR168 Evades Sequestration by the Tombusviral P19 VSR to form Complexes with AGO10. (A) P19 effects on ArabidopsisAGO1 binding to miRNAs. Crude extracts were prepared from leaves of SUC-SUL (SS) plants or SS plants expressing FLAG-HA-tagged TBSV P19 (SS + P19FH). AGO1 was immunopurified with anti-AGO1 antibody (control IP w/o antibody). P19FH levels were analyzed with anti-HA antibody. CBB staining serves as a loading control. Steady-state and AGO1-bound levels of miR168, miR162, and miR159 were determined by RNA blot analysis. (B and C) sRNA duplex gel-mobility shift induced by P19 in vitro. TBSV P19 was expressed in BYL by in vitro translation and mixed with mock BYL to prepare conditions of increasing P19 levels (%). The mixtures were incubated with 32P-labeled 21-nt siRNA, miR168, or miR162 duplexes (B), and 21-nt WT or m1-4 miR168 duplexes (C). Signal intensities of free and P19-associated duplexes were quantified to measure the proportion bound to P19. (D) P19 effects on miR168 loading into Arabidopsis AGO1 or AGO10 expressed in N. benthamiana leaves. In the left panel, WT or m4 pri-miR168a (control: buffer w/o pri-miR168a) was expressed together with GFP-tagged AGO1 or AGO10 (control: buffer w/o AGO). AGO immunopurification was performed with anti-GFP antibody. miR168 levels in input and AGO-bound fractions (GFP-IP) were determined by RNA blot analysis. In the right panel, miR168 and AGO expression and GFP-IP were performed with co-expressed TBSV P19FH. Molecular Plant 2018 11, 1008-1023DOI: (10.1016/j.molp.2018.05.006) Copyright © 2018 The Author Terms and Conditions

Figure 7 A Model for miR168 Functional Versatility Induced by Duplex Structure Flexibility and the Impact of VSR P19. Using the flexible motif, miR168a precursors effectively alter the duplex structures (states I, II, and III). The stabilized state-III alters the DCL1 cleavage position on pre-miR168a, generating 22-nt miR168a duplexes. These state-III-derived 22-nt miR168 duplexes are potent transitivity inducers and exhibit high affinity for AGO10. In the presence of tombusvirus P19, the state-III duplexes are not sequestered by P19, unlike other vsiRNA- and abundant miRNA -duplexes, thereby facilitating their loading into AGO10. The P19-induced miR168-AGO10 RISC dampens AGO1 levels and activity via translational repression. Molecular Plant 2018 11, 1008-1023DOI: (10.1016/j.molp.2018.05.006) Copyright © 2018 The Author Terms and Conditions