MicroRNAs (miRNAs) regulate the levels of translation of messenger RNAs (mRNAs). At present, the major parameter that can explain the selection of the target mRNA and the efficiency of translation repression is the base pairing between the ‘seed’ region of the miRNA and its counterpart mRNA1. Here we use R1ρ relaxation-dispersion nuclear magnetic resonance2 and molecular simulations3 to reveal a dynamic switch—based on the rearrangement of a single base pair in the miRNA–mRNA duplex—that elongates a weak five-base-pair seed to a complete seven-base-pair seed. This switch also causes coaxial stacking of the seed and supplementary helix fitting into human Argonaute 2 protein (Ago2), reminiscent of an active state in prokaryotic Ago4,5. Stabilizing this transient state leads to enhanced repression of the target mRNA in cells, revealing the importance of this miRNA–mRNA structure. Our observations tie together previous findings regarding the stepwise miRNA targeting process from an initial ‘screening’ state to an ‘active’ state, and unveil the role of the RNA duplex beyond the seed in Ago2.
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NMR sequence-specific resonance assignments have been deposited in the Biological Magnetic Resonance Data Bank under accesssion numbers 27225 (hsa-miR-34a-5p), 27226 (the miR34a–mSirt1 bulge) and 27229 (the miR34a–mSirt1 trapped excited state). The plasmids used for the DLR assay were a gift from J. Weidhaas (Addgene plasmids 78258 and 78259). All data and code used for data analysis are available upon request. The ensembles of REMD simulations have been deposited in Model Archive (www.modelarchive.org) under accession numbers ma-bc9uo, ma-z54y4 and ma-g8e5z.
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We thank the members of the Petzold and the E. R. Andersson laboratories for insightful discussions; N. H. Hopkins for providing the curated chemical shifts and for contributing to the MC-Fold secondary-structure-prediction script; I. MacRae for the gift of plasmid expressing human Ago2; and the Lund University Protein Production Platform (LP3) (Lund, Sweden) and the Protein Science Facility at the Karolinska Institute Department of Medical Biochemistry and Biophysics (Stockholm, Sweden) for help with human Ago2. K.P. acknowledges funding from the Swedish Research Council (grant number 2014-04303), the Swedish Foundation for Strategic Research (project number ICA14-0023), Harald och Greta Jeansson Stiftelse (JS20140009), Carl Tryggers stiftelse (CTS14-383 and 15-383), Eva och Oscar Ahréns Stiftelse, Åke Wiberg Stiftelse (467080968 and M14-0109), Cancerfonden (CAN 2015/388), the Karolinska Institute Department of Medical Biochemistry and Biophysics (grant number KID 2-3707/2013 and support for the purchase of a 600-MHz Bruker NMR spectrometer) and Ragnar Söderberg Stiftelse (M91/14). A.A.C. acknowledges support from National Science Foundation (grant MCB1651877). B.F. acknowledges funding from the Strategic Research Area (SFO) program of the Swedish Research Council (VR) through Stockholm University. This work used the computational resources of the Extreme Science and Engineering Discovery Environment (XSEDE) (allocation TG-MCB140273 to A.A.C.), which is supported by National Science Foundation grant number ACI-1548562. J.S. acknowledges funding through a Marie Sklodowska-Curie Individual Fellowship (EU H2020/project number 747446).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Secondary-structure rearrangements among the ten lowest-energy structures were calculated using MC-Fold21. Ranking (numbers in parentheses) according to the predicted energy difference, based on the minimum free energy (MFE), is indicated in each label (ΔΔG(n) in units of unreferenced kcal mol−1, as described21). Secondary structures with a single base-pair opening in the cUUCGg region are omitted. a, The miR-34a–mSirt1 duplex connected by a cUUCGg loop (black). The MFE corresponds to a 7-mer–A1 binding site. Suboptimal structures (3) and (5) suggest possible modulation of the binding site to a 8-mer–GU and an 6-mer–A1 configuration, respectively. b, miR-34a–mSirt1 bulge construct, comprising a cUUCGg loop and a closing stem (black). The secondary-structure distribution of the miR-34a–mSirt1 bulge follows the same trend as the full-length duplex; dashed lines connect identical bulge structures. Suboptimal structures were used to validate or reject models of excited-state (ES) secondary structures on the basis of R1ρ NMR relaxation-dispersion data. Structure (1), with the MFE, corresponds to the assigned ground-state structure (GS). Structure (3) satisfies the 1H1 and 15N1 R1ρ NMR relaxation-dispersion data on gG6(G24), being G:U base paired with tU20(U9). Structure (5) is mutually exclusive with (3) in structural terms and satisfies the 13C R1ρ NMR relaxation-dispersion data measured on tA19 that indicate this residue adopting a base-paired conformation. Therefore we propose structure (3) as ES1 and structure (5) as ES2. Conformations (6) and (7) do not agree, and partially clash, with our R1ρ NMR relaxation-dispersion data and can therefore be excluded as excited states. c, miR-34a–mSirt1 trapped excited-state duplex connected by a cUUCGg loop (black). Substituted nucleotides used to trap the excited state are in yellow. The MFE corresponds to a 8-mer binding site. d, miR-34a–mSirt1 (turquoise) trapped excited-state construct comprising an cUUCGg loop and a closing stem (black). Substituted nucleotides used to trap the excited state are in yellow. The secondary-structure distribution of the miR-34a–mSirt1 trapped excited state follows a similar trend as the full-length duplex; dashed lines connect identical bulge structures.
Shown are HSQC overlays of different Mg2+ titration steps. a, 1H–13C aromatic 2/6/8 HSQC. b, 1H–13C sugar 1′ HSQC. c, 1H–15N imino 1/3 HSQC. The titration steps are colour-coded (a, top left). Additional overlay of the miR-34a–mSirt1 trapped ES is shown in grey in a, b. Arrows indicate the chemical-shift trajectory during titration. Dashed lines connect equivalent peaks in the miR-34a–mSirt1 bulge and trapped ES constructs.
Extended Data Fig. 3 MCSF analysis of the miR-34a–mSIRT1 bulge and trapped excited state, and analysis of 13C tA22C8 outliers.
a, b, We used the MCSF approach22 to cross-validate our candidate excited state (ES1), modelled using R1ρ-derived ground-state-to-excited-state chemical-shift differences (a, 13C R1ρ Δω data, blue dots; b, left). pES refers to the excited-state population (popES in the main text). We also generated an ES1 mimic (trapped ES1) using a two-point substitution, predicted to stabilize the proposed conformation (b, bottom). For each reporter atom, we compared 13C R1ρ Δω data with the chemical-shift differences derived from the assignment of the bulge and the trapped ES constructs (a, 13C Δω trapped ES (tES) data, turquoise dots). In a, The MCSF analysis validates our ES1 model (green shading), with exceptions arising from the limitations of the mimic (orange shading) and from the presence (violet shading) of a second ES (ES2, b, right). Errors for R1ρ relaxation-dispersion-derived Δω represent 1 s.d. from fitting (see also Supplementary Methods). In b, the proposed model for ES2 satisfies the 13C R1ρ Δω data measured for tA19 and gG6. GS, ground state. c, The free-energy landscape for the entire star-like three-state exchange process. (The MCSF analysis and ES2 are discussed further in the Supplementary Information, Discussion 5.) The transition coeffient (κ), is assumed to be 1 (ref. 23), so the transition-state energies (TS1 and TS2), calculated using Supplementary equation (11), must be considered an upper limit of this exchange process. d, e, The substitution site (tU21 to tC21) perturbs the chemical environment of tA22C8 that is directly neighbouring the substituted nucleobase (orange sphere in e). Conversely, tA22C2 (green sphere), pointing towards the miR-34a strand (red), experiences an equivalent chemical environment in the bulge (blue) and trapped ES (turquoise) constructs. This explains the inconsistency in the MCSF profile for tA22C8 (Supplementary Fig. 12a, orange box). d, Secondary structure environment of tA22 in the miR-34a–mSirt1 bulge excited state (left) and trapped ES (right) constructs. The substitution site (tU21 to tC21) is highlighted. e, Overlay of average structures of the bulge ES (blue) and trapped ES (turquoise) from REMD ensembles, aligned according to residues gU7 and tA22. Residues gU7, gG8, tU21 and tA22 are shown. tA22C8 and tA22C2 13C atoms are in orange and green respectively.
a, Individual A260 UV melting profiles for the constructs used here. The miR-34a–mSirt1 duplex, miR-34a–mSirt1 trapped ES duplex, miR-34a–complementary-strand duplex and miR-34a single-stranded RNA (ssRNA) were each measured as three technical independent replicates (shown in different colours; n = 1). Individual technical replicates are plotted. Tm values are shown as means ± s.d. of fitted Tm values in individual technical replicates (n = 3). The other ssRNAs (bottom row) were measured and plotted as individual technical replicates; fitted Tm values are shown with associated confidence intervals of 95% (n = 1) as an estimate of the experimental error. Normalized differential melting curves (δA260/δT) are plotted as a function of temperature (in K) (circles) and fitted to Supplementary equation (1a) or (1b) (curves), depending on the molecularity of the system. b, EMSA titration profiles for the miR-34a–mSirt1 duplex, miR-34a–mSirt1 trapped ES duplex and miR-34a–complementary-strand duplex, measured as three independent technical replicates. The ratio of bound to total miR-34a 3′-Cy3 is plotted as a function of titrand concentration (circles) and fits a standard binding isotherm (line) (Supplementary equation (2)). The plot centre is the mean; error bars represent 1 s.d. from the three independent replicates. Fitted Kd values along with confidence intervals of 95% are shown as an estimate of the experimental error (n = 3). Gel images were acquired by detection of Cy3 fluorescence. During the titration, miR-34a 3′-Cy3 was kept at a constant concentration of 24 nM, setting the sensitivity limit for estimating Kd (Supplementary Fig. 1a–c). mSirt1 and its trapped-ES counterpart are equivalent in their ability to form a stable RNA–RNA duplex with miR-34a. Tighter binding is observed for the complementary strand (48.4 ± 9.5 nM) than for the mSirt1 (124.3 ± 21.7 nM) and trapped-ES mSirt1 (110.3 ± 23.0 nM), providing a control for the dynamic range of Kd estimation. c, Equilibrium FBA profiles for mSirt1, mSirt1 trapped ES and a scrambled control, binding to miR-34a-loaded Ago2. The three targets were each measured as three independent replicates and fitted to a standard binding isotherm (line) (Supplementary equation (2)). The plot centre is the mean; error bars represent 1 s.d. from three independent replicates. Fitted Kd values are shown with confidence intervals of 95% (an estimate of the experimental error). As in c, mSirt1 and mSirt1 trapped ES are equivalent in their ability to form a stable ternary complex within RISC. The simulated data set (dotted lines) indicate curves corresponding to Kd values ten times lower (red) or ten times higher (green) than the average value for mSirt1 and mSirt1 trapped ES, providing a frame for the amplitude of our experimental error. d, Top, northern blot showing the detection of miR-34a loaded in Ago2. Bottom, a standard calibration curve (using naked miR-34a), used to obtain an estimate of miR-34a in RISC. The centre calibration curve was used to calculate R2. The two outer curves indicate the 95% confidence interval of the calibration-line fit (from a single repeated experiment). The average ratio of Ago2 and miR-34a-loaded Ago2 (both in pmole) was used to obtain the fraction of Ago2 loaded with our guide (roughly 1.5%). The complete lists of fitted parameters for UV melting, EMSA titration, FBA titration and northern blot are in Supplementary Table 1a–d. The complete fitting analyses of UV melting, EMSA titration and FBA titration are in Supplementary Tables 7–9.
Superposition of ground state (green) and excited state (orange) conformational ensembles on the Ago2 crystal structure (PDB code 4W5T), with seed sequences aligned to crystallographic miRNA–mRNA positions (red). Although the seed orientations are comparable, the ground-state and excited-state conformations sample different space within Ago2.
Extended Data Fig. 6 Slow-growth insertion of excited-state RNA into Ago2 predicts the ability of bulged miRNA–mRNA complexes to access an alternative dsRNA-binding mode of Ago2.
Slow-growth induced-fit Ago2 structures are compared with existing X-ray structures (whose PDB identification codes are shown in the figure) via structural alignment. a, Ago2 after induced fit with ES RNA binds in the PIWI-adjacent groove rather than the PAZ domain. b, The Thermus thermophilus (Tt) Ago crystal structure similarly shows DNA/RNA-duplex binding in the analogous PIWI-adjacent groove. c–j, The root mean square deviation (r.m.s.d.) for each indicated pair of Ago structures was measured after structural alignment either of all protein atoms, or excluding the PAZ domain, PIWI loops and helix-7 atoms (‘subset aligned’; these excluded atoms still count towards the r.m.s.d.). The subset-aligned structures show that most of the r.m.s.d. difference arises from pivoting motions of the PAZ domain, coupled with small shifts in helix-7 and PIWI loops to accommodate the inserted ES RNA structures. c, Comparison of slow-growth human Ago2–GS and the existing Ago2 structure (PDB code 4OLA; r.m.s.d. = 2.065 Å (all) and 2.62 Å (subset aligned)). d, Comparison of slow-growth Ago2–ES and the 4OLA structure (r.m.s.d. = 1.4 Å (all) and 1.65 Å (subset aligned)). e, Comparison of the slow-growth Ago2–trapped ES and the 4OLA structure (r.m.s.d. = 1.9 Å (all) and 2.18 Å (subset aligned)). f, Comparison of the slow-growth Ago2–GS with Ago2–ES (r.m.s.d. = 2.1 Å (all) and 2.2 Å (subset aligned)). g, Comparison of the slow-growth Ago2–ES with Ago2–trapped ES (r.m.s.d. = 1.6 Å (all) and 1.33 Å (subset aligned)). h, Comparison of the slow-growth Ago2–GS and the 6N4O structure (r.m.s.d. = 2.05 Å (all) and 2.065 Å (subset aligned)). i, Comparison of the slow-growth Ago2–ES (green) with the 3HM9 structure (r.m.s.d. = 4.52 Å (all)). j, Comparison of the slow-growth Ago2–GS with the 3HM9 structure (r.m.s.d. = 3.85Å (all)).
We studied five targets of different bulge sizes (see Methods). Individual replicates are plotted as circles; the centre line is the mean; error bars represent 1 s.d from three independent replicates; nts, nucleotides. a, Standard DLR normalization (relative to the control condition with no miR-34a duplex transfected). Despite the large variability between replicates, a consistent increase in downregulation (connecting lines) is observed for wild-type (WT) and trapped excited-state (tES) constructs. b, When the data sets are internally normalized and the WT condition is set to 100% (mean value), the variation due to experimental replicas is attenuated and the trend observed in a is maintained.
This file contains the Supplementary Discussion, Supplementary Methods, Supplementary Figures 1-9, a full description of the Supplementary Data (Supplementary Data file supplied separately) and Supplementary References.
This file contains the uncropped gels.
This file contains fitting details and materials – see Supplementary Information document for full description.
Morphing transition between GS-ES Ago2 structures shows that the accommodation of the ES state requires pivoting of the PAZ domain.
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Baronti, L., Guzzetti, I., Ebrahimi, P. et al. Base-pair conformational switch modulates miR-34a targeting of Sirt1 mRNA. Nature 583, 139–144 (2020). https://doi.org/10.1038/s41586-020-2336-3
Nature Communications (2022)
Nature Communications (2021)
Mutate-and-chemical-shift-fingerprint (MCSF) to characterize excited states in RNA using NMR spectroscopy
Nature Protocols (2021)
Communications Biology (2021)
Nature Structural & Molecular Biology (2020)