1. Molecular Insights into microRNA-Mediated Translational Repression in Plants 
Hiro-oki Iwakawa, Yukihide Tomari 
Molecular Cell 
Volume 52, Issue 4, Pages (November 2013)
DOI: /j.molcel
Copyright © 2013 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2013 52, 591-601DOI: (10.1016/j.molcel.2013.10.033)
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 1 AtAGO1-RISC Mediates Translational Repression in Plant Lysate
(A) Scheme for the experimental procedure.
(B) Schematic representation of Rluc reporter mRNAs carrying one, two, four, and eight copies of the target sites (black boxes) in the 3′ UTR (3′ UTR-1×–UTR-8×). The base-pairing configurations between the target sequence and the guide strands of the two small RNAs used in (C–F) are shown below.
(C and E) Integrity of Rluc target mRNAs and control Fluc mRNAs in the presence of WT (C) or catalytic mutant (E) AtAGO1-RISC. No small RNAs were added in the control (mock). The target mRNAs remained intact except for the combination of WT AtAGO1-RISC and perfect complementarity. Arrowheads indicate 5′ cleavage products.
(D and F) Translational repression by WT (D) or catalytic mutant (F) AtAGO1-RISC. AtAGO1-RISC repressed the translation of target mRNAs in a manner dependent on the number of target sites at least up to four. The Rluc/Fluc luminescence was normalized to the value of no small RNA (mock). The mean values ± SD from three independent experiments are shown.
(G) Schematic representation of the reporter mRNAs with target sites (black boxes) in different positions. The base pairing configurations between the target sequence and the guide strands of the five small RNAs used in (H–K) are shown below.
(H and I) Integrity of Rluc target mRNAs and control Fluc mRNAs in the presence of WT (H) or catalytic mutant (I) AtAGO1-RISC. No small RNA was added in the control (mock). The target mRNAs remained intact except for the combination of WT AtAGO1-RISC and perfect complementarity. Arrowheads indicate 5′ cleavage products.
(J and K) Translational repression by WT (J) or catalytic mutant (K) AtAGO1-RISC. Both complementarity and the positions of the target sites affected the degree of translational repression. The Rluc/Fluc luminescence was normalized to the value of no small RNA (mock). The mean values ± SD from three independent experiments are shown.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

4. Figure 2
AtAGO1-RISC Blocks Translation via Different Mechanisms Depending on the Position of Target Sites
(A) Scheme for the experimental procedure.
(B) Schematic representation of the cap-radiolabeled reporter mRNAs carrying a short ORF and a target site (black box) in the 5′ UTR (short-5′ UTR), ORF (short-ORF), or 3′ UTR (short-3′ UTR-1×) or eight target sites in the 3′ UTR (short-3′ UTR-8×).
(C–F) Sucrose gradient analysis of cap-radiolabeled short-5′ UTR (C), short-ORF (D), short-3′ UTR-1× (E), and short-3′ UTR-8× (F) in the presence (red line) or absence (black line) of catalytic mutant AtAGO1-RISC with perfect complementarity to the target sequence. AtAGO1-RISC repressed the formation of the 80S initiation complex. The peaks detected in the light fractions (fraction numbers 1–7 [C–F]) show the ribosome-free target mRNAs or RNPs. The mean values ± SD from three independent experiments are shown.
(G) Schematic representation of the A-capped Rluc reporter mRNAs carrying TEV IRES and a target site (black box) in the 5′ UTR (TEV IRES-5′ UTR), ORF (TEV IRES-ORF), or 3′ UTR (TEV IRES-3′ UTR-1×) or eight target sites in the 3′ UTR (TEV IRES-3′ UTR-8×).
(H and I) Translational repression by catalytic mutant AtAGO1-RISC for m7G-capped target mRNAs (H) or A-capped TEV IRES target mRNAs (I). The Rluc/Fluc luminescence was normalized to the value of no small RNA (mock). Cap-dependent translation was repressed via 5′ UTR, ORF, and 3′ UTR target sites (H). In contrast, TEV-IRES-mediated translation was refractory to translational repression via the 3′ UTR target sites. The mean values ± SD from three independent experiments are shown.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

5. Figure 3 AtAGO1-RISC Bound to the ORF Blocks Translation Elongation
(A) Schematic representation of the Rluc reporter mRNAs carrying N-terminal 3× FLAG tag (gray box) and a perfectly complementary target site (black box) in the 5′ UTR (FLAG-5′ UTR) or at different positions within the ORF (FLAG-ORF-1 and FLAG-ORF-2). The base pairing configurations between the target sequence and the guide strands of the two small RNAs used in (B) are shown below.
(B) Northern blotting of the Rluc reporter mRNAs (top) and anti-FLAG western blotting of the reporter products (middle and bottom) under translational repression by catalytic mutant AtAGO1-RISC programmed with small RNAs shown in (A) and Table S1. Truncated proteins (arrowheads) were accumulated when AtAGO1-RISC bound to the ORF sites with perfect complementarity.
(C) Schematic representation of the natural miRNA target mRNAs carrying N-terminal 3× FLAG-tagged (gray box) CSD1 and SPL13 and the base-pairing configurations between the target sequence and the guide strands of miR398 and miR156.
(D and E) Northern blotting of the natural target mRNAs (top) and anti-FLAG western blotting of the reporter products (bottom) in the presence or absence of WT or catalytic mutant AtAGO1-RISC programmed with cognate miRNAs. Truncated proteins were accumulated when AtAGO1-RISC targeted the SPL13 mRNA.
(F) A parallel time-course analysis of protein synthesis and mRNA stability of the SPL13 target in the presence or absence of WT catalytically active AtAGO1-RISC. Although substantial fractions of full-length mRNAs remained uncleaved, full-length protein was barely produced.
See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

6. Figure 4 Target Binding by AtAGO1-RISC
(A) Schematic representation of the base-pairing configuration between the 2′-O-methylated 21 nt ASO and the target sequence.
(B) Translational repression by catalytic mutant AtAGO1-RISC or ASO with perfect complementarity to the target sequence via the 5′ UTR (top) or ORF (bottom) target site. AtAGO1-RISC repressed translation far more strongly than ASO. The mean values ± SD from three independent experiments are shown.
(C) Translational repression by ASO preannealed with the target mRNAs. The mean values ± SD from three independent experiments are shown.
(D) Scheme for the purification of catalytic mutant AtAGO1-RISC.
(E) The base-pairing configurations between the target sequence and the guide strands of the five small RNAs used in (F and G).
(F) Equilibrium binding assay with purified catalytic mutant AtAGO1-RISC and targets shown in (E). AtAGO1-RISC requires extensive complementarity for target binding. The mean values ± SD from four independent experiments are shown.
(G) Dissociation rate for a fully complementary, 2 and 4 nt centrally mismatched target RNAs. AtAGO1-RISC slowly binds to and dissociates from perfectly complementary sites. The mean values ± SD from three independent experiments are shown.
(H) A model of translational repression in plans. When extensively complementary target sites reside in the 5′ UTR or ORF, AtAGO1-RISC can sterically hinder ribosomal recruitment or movement. In contrast, when there are multiple target sites in the 3′ UTR, AtAGO-1 RISC can mediate the animal-like repression of translation initiation.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions