Analysis of MicroRNA-Induced Silencing Complex-Involved MicroRNA

Article pubs.acs.org/ac

Analysis of MicroRNA-Induced Silencing Complex-Involved MicroRNA-Target Recognition by Single-Molecule Fluorescence Resonance Energy Transfer Ying Li and Chun-yang Zhang* Single-molecule Detection and Imaging Laboratory, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Guangdong, 518055, China S Supporting Information *

ABSTRACT: MicroRNAs (miRNAs) are important regulators of gene expression that control almost every physiological and pathological process. Although the complementarity between the seed region of a miRNA and its target mRNA is usually deemed as the key determinant in the miRNA-target recognition in animals, the mechanism of their recognition still remains enigmatic as more and more exceptions challenge the seed rule. Herein, we employ single-molecule fluorescence resonance energy transfer (smFRET) to investigate human miRNA-induced silencing complex (miRISC)-involved miRNA-target recognition with either perfect base pairing or poor seed match in real time. Our results demonstrate that the recognition between mammalian miRNA and its target with perfect base pairing proceeds in a two-state model as prokaryotic guide DNA-mediated recognition, suggesting a conserved pattern of guide RNA/DNA strand recognition. In addition to the general rule of miRNA-target recognition, our results reveal that annealing between miRNA and its target with poor seed match proceeds in a stepwise way, which is in accordance with the increase in the number of conformational states of miRNA-target duplex accommodated by the miRISC, suggesting the structural plasticity of human miRISC to conciliate the mismatches in seed region. This new dynamic information revealed by smFRET has an important implication for comprehensive understanding of the role of miRISC in the target recognition in mammals. icroRNAs (miRNAs) comprise a large family of ∼22-nt noncoding RNAs that have emerged as key posttranscriptional regulators of gene expression in metazoan animals and plants.1 Generally, miRNAs inhibit protein synthesis by repressing translation or inducing mRNA decay.2 In mammals, miRNAs control the activity of more than 60% of all proteincoding genes3 and participate in the regulation of cell differentiation, proliferation, apoptosis, and metabolism. Further research demonstrates that the changes in the expression of miRNAs are associated with various pathologies.4−6 miRNAs function in the form of miRNA-induced silencing complexes (miRISC). Human miRISC is assumed to be composed of Argonaute, RNA-generating enzyme Dicer, and TRBP (trans-activation response RNA-binding protein).7,8 However, the validated constitution of human miRISC-loading complex remains under debate.9 Several regulatory factors, such as GW182 (glycine-tryptophan protein of 182 kDa),10 the fragile X mental retardation protein (FMRP),11 and endoribonuclease C3PO (component 3 promoter of RISC),12 are also involved in modulating miRNA function. At a molecular level, miRNAs guide the miRISC to their mRNA targets through perfect or partial matching following the base-pairing rules.13 It has been known that plant miRNAs often have targets with perfect or near-perfect complementarity, facilitating

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© 2012 American Chemical Society

relatively simple identification. In contrast, it is rare for animal miRNAs to identify targets with perfect complementarity. Target recognition is mainly based on contiguous Watson− Crick pairing centered in the miRNA positions (the so-called seed sequence).14 However, this miRNA target code is expanded by 3′ compensatory pairing and centered pairing.15,16 Intriguingly, some uncanonical base pairings with poor seed match are experimentally identified in the mammals.17,18 Since sequence parameters alone are insufficient to determine target sites, the miRISC onto which the miRNA is loaded might be a key factor that influences the target recognition.17 Recently, a series of X-ray crystallography studies, which captured and visualized Argonaute from Thermus thermophilus with guide DNA and complementary target RNA strands, provided a structural view on the formation of prokaryotic guide−target duplex.19−21 However, the dynamic recognition between miRNA and the target in mammalian miRISC has never been described yet, especially for the “seedless” case. Single-molecule fluorescence resonance energy transfer (smFRET) has been successfully used to characterize the conformational fluctuations of single biological molecules that Received: March 27, 2012 Accepted: April 30, 2012 Published: April 30, 2012 5097

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Preparation of Cell Lysate. HeLa cell lysate was prepared as previously described.9 Briefly, HeLa cells were washed twice with precold PBS (pH 7.4) and collected by centrifugation at 1000g. The cell pellet was resuspended in a 2-pellet volume of lysis buffer (30 mM HEPES-KOH (pH 7.4), 100 mM potassium acetate, and 2 mM magnesium acetate), containing 5 mM DTT and protease inhibitor cocktails (Sigma), and homogenized with a 25-G needle (50 strokes). The lysate was clarified by centrifugation at 17 000g at 4 °C for 20 min. The supernatant was flash frozen in liquid nitrogen and stored at −80 °C. Binding and Cleavage Assays. The binding affinity of miRISC to miRNA was determined as previously described.27,28 ATTO565-labeled miRNAs (0.1 μM) were incubated with 10 μL of 4 μg/μL HeLa cell lysate in a 50 μL reaction system (10 mM Tris, pH 7.4, 100 mM KCl, 2 mM MgCl2, 20 units of RNasin, and 1 mM DTT) at 30 °C for 1 h. Ethylenediaminetetraacetic acid (EDTA, 7 mM) was used instead of 2 mM MgCl2 in the Mg2+-free reaction. Binding reactions were terminated on ice. After adding 5 μL of loading buffer (50% (v/v) glycerol, 1% (w/v) bromophenol blue, and 1% (w/ v) xylene cyanol), 30 μL aliquots were electrophoresed by 6% native polyacrylamide gel electrophoresis (PAGE) at 100 V for 1 h on ice. The gels were imaged by Kodak 4000MM with 535 nm (±15 nm) excitation filter and 600 nm (±17.5 nm) emission filter. The target cleavage assays were performed in a 20 μL reaction system. Synthesized miRNA (25 nM) was preincubated with 2 μL of HeLa lysate for 30 min, followed by incubation with 5 nM target at 30 °C for 2 h and then diluted 25 times for smFRET measurement. Western Blotting Assay. HeLa cell lysate was quantified using BCA protein assay kit (Merck). Equal amounts of protein were resolved by sodium dodecyl sulfate (SDS)-PAGE on 10% gels. Anti-human Ago2 (Cell Signaling Technology, 1:1000) and anti-α tublin (Santa Cruz, 1:1000) were used as the primary antibodies. Chemiluminescence was induced by electrochemiluminescence (ECL) Western blotting substrate (Pierce). The images were acquired by Kodak 4000MM and quantified with ImageJ software.

are difficult to synchronize or too rare to be detected by the ensemble studies.22 Herein, we employ smFRET to detect the detailed kinetics of miRISC-involved miRNA-target recognition in real time. Our research provides a dynamic perspective for the miRNA-target recognition and reveals the two-state model of miRNA-target recognition as well as the capability of human miRISC to conciliate the mismatches in the seed region.

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EXPERIMENTAL SECTION Materials. All oligonucleotides were purchased from BioSynthesis Inc. (Lewisville, TX). The sequence of both perfect match and seedless match groups were designed according to the published reports.18,23 The exact labeling positions of ATTO565 in the miRNA and ATTO647 in the target were chosen to span the duplex forming fragment (corresponding to positions 2−16 in the miRNA) as far as possible, with compulsory modifications due to the limit of dye conjugation. The sequence and modifications are as follows: let-7a-3, 5′-PUGA GGU AGU AGG UUG UATTO565AU AGU U-3′; target of let-7a-3, 5′-biotin-GGU AUC AAC CAC UAU ACA ACC UAC UAC CUATTO647C AAC GUU CA-3′; miR-24, 5′-P-UGG CUC AGU UATTO565CA GCA GGA ACA G-3′; target of miR-24, 5′biotin-UAA GCA ACU GGA UCA AUU UGC UGA CUU GGG CAUATTO647 AAU CUA AUC-3′. Single-Molecule FRET Measurement. Target oligonucleotides were immobilized on the PEG-coated quartz slides through biotin−streptavidin linkage as described previously.24 ATTO565 (donor) was excited by a Jive 561-nm DPSS laser (Cobolt) via total internal reflection. The fluorescence signals from both the donor and the acceptor were collected by an oil immersion objective (NA 1.45, 100×, Olympus), separated by Optosplit II image splitter (Cairn Research), and imaged onto the two halves of an Andor Ixon DU897 EMCCD with a time resolution of 50 ms. Measurements were performed at 25 °C for Figure 2 and at 30 °C for Figures 3 and 4 in the buffer of 10 mM Tris, pH 7.4, 100 mM KCl, 2 mM MgCl2, 20 units of RNasin, and 1 mM dithiothreitol (DTT).25 An oxygen scavenger system (10% w/v glucose, 300 μg/mL glucose oxidase, and 40 μg/mL catalase, 2 mM Trolox) was also included in the imaging buffer to minimize the photobleaching. During the dynamic image acquisition, the imaging chamber (Warner Instruments) was preincubated with 200 pM targets before the addition of miRNA (25 nM) alone or preincubation with Hela cell lysate. Data analysis of the fluorescence time traces were performed using the software shared by Dr. Ha.24 FRET efficiency was calculated as IA/(IA + ID), where IA and ID were the fluorescence signals from the acceptor and the donor, respectively. The stepwise increase in FRET observed in miRNA winding was processed via Matlab program developed by Dr. Gonzalez Jr. et al.26 Cell Culture and Knockdown. HeLa cells were cultured in Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% FBS, 100 μg/mL penicillin, and 100 μg/mL streptomycin. Cells were incubated at 37 °C in humidified air with 5% CO2 and subcultured every 2 or 3 days. Ago2 knockdown in HeLa cells was performed by transfecting a siRNA duplex (siAgo2, 5′-CGA UCG GCA AGA AGA GAU UAG-3′ and 5′-AAU CUC UUC UUG CCG AUC GGG-3′)27 using Lipofectamine 2000 (Invitrogen). One day before transfection, cells were plated in 6 cm dishes without antibiotics, transfected with 300 pM siRNA when cells were 70% confluent, and harvested after 48 h.

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RESULTS AND DISCUSSION Two experimental schemes were designed: one had perfect match between miRNA and miRNA recognition element (MRE) (perfect match group with let-7a-3 and MRE, Scheme 1B);23 the other conducted a “seedless” recognition model (seedless match group with miR-24 and MRE, Scheme 1C).18 The selected target fragment containing MRE from the fulllength 3′ untranslated regions (3′ UTRs) complied with the rule of site accessibility for target recognition (see the Supporting Information, Table S1).29 To obtain a FRET signature suitable for discriminating individual steps during miRNA-target annealing, the labeling positions of ATTO565 (donor) in the miRNA and ATTO647 (acceptor) in MRE were chosen to span the duplex forming fragment (corresponding to the positions 2−16 in miRNA) based on the structural analysis of TtAgo containing the guide and the target.21 Mammalian miRNA-target interaction occurred in the context of multicomponent miRISC in vivo,7−12 but it was difficult to reconstitute such multicomponent system in vitro. Alternatively, we obtained the native human miRISC system by the use of whole HeLa cell lysate.9 To confirm that the HeLa cell lysate possessed native bioactivity of miRISC, ATTO565labeled miRNA was incubated with HeLa cell lysate and 5098

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Scheme 1. Scheme for Single-Molecule Analysis of miRISCInvolved miRNA-Target Recognitiona

Figure 1. Assembly of miRNA into the miRISC in HeLa lysate. (A) The assembly of miRNA into the miRISC was dependent on Ago2. Wild-type (Mock) and two copies of Ago2-silenced HeLa cell lysate were incubated with 0.1 μM synthetic 5′ P-miR-24, followed by native PAGE analysis. Synthetic miRNAs with same amount were used as the markers. Western blot of human Ago2 from the corresponding total HeLa cell lysate was shown below. α-Tubulin was used as the control. (B) Assembly of miRNA into the miRISC was dependent on Mg2+. Native PAGE analysis of miRISC assembly was performed in the presence of either 2 mM Mg2+ or 7 mM EDTA. Synthetic miRNAs with same amount were used as the markers. (C) The normalized percentage of free miRNAs from the corresponding lanes in B.

a

(A) Experimental scheme. Target oligonucleotides were immobilized on a polymer-coated quartz surface by the biotin−streptavidin interaction. (B) Perfect match between the miRNA and MRE. (C) Seedless match between the miRNA and MRE. The miRNAs were highlighted in blue. The connecting solid lines indicated a Watson− Crick base pair, and the connecting interrupted lines indicated a GU wobble pair. The mismatch positions between the donor (green) and the acceptor (red) were shadowed in gray.

detected by the electrophoretic mobility shift assay. Contrary to the miRNA alone (line 4 in Figure 1A), most miRNA which preincubated with HeLa cell lysate presented in the form of miRISC and migrated more slowly (Line 1 in Figure 1A) than free miRNA molecules. Moreover, consistent with the fact that Ago2 directly interacted with the miRNA in miRISC,19,20 inhibition of endogenous expression of Ago2 by RNAi increased the amount of unassembled miRNA (lines 2 and 3 in Figure 1A). To further validate the native bioactivity of miRISC in Hela cell lysate, the assembly of miRNA into the miRISC was evaluated in the presence and in the absence of Mg2+, respectively. In the presence of Mg2+, about 80% miRNAs were assembled into silence complex (line 2 and line 5 in Figure 1B,C), whereas a significant increase in the amount of free miRNAs was observed with the depletion of Mg2+ from the reaction buffer by EDTA (line 3 and line 6 in Figure 1B,C). This was in agreement with the research on crystal structures of archaeal Pyrococcus f uriosus Ago which contained two Mg2+binding sites: one was positioned at the catalytic pocket, and the other was positioned at the basic binding pocket, which was favorable to interact with the 5′ terminus of the antisense RNA.30 In the following study, smFRET was applied to probe the miRNA-target recognition either in the absence or in the presence of Hela cell lysate by monitoring the fluorescence signals of the donor and the acceptor obtained from the totalinternal-reflection fluorescence microscope. Figure 2A,B shows the FRET histograms obtained from the perfect match group (let-7a-3 and target) in the absence and in the presence of HeLa cell lysate, respectively. Two populations of FRET efficiency emerged in the absence of HeLa cell lysate (Figure 2A): the low efficiency subpopulation representing the

immobilized targets alone (E = 0.13) and the other corresponding to the annealed RNA duplex (E = 0.32). After incubation with the HeLa cell lysate, the low-FRET peak (E = 0.13) became a dominant state (Figure 2B), indicating the cleavage of the targets by Ago2 in the miRISC as a result of extensive base pairing of miRNA-target.25 Figure 2C,D shows the FRET histograms obtained from the seedless match group (miR-24 and the target) in the absence and in the presence of HeLa cell lysate, respectively. Only a single narrow peak at 0.15 was observed in the absence of HeLa cell lysate (Figure 2C). Remarkably, upon the addition of HeLa cell lysate, four small yet noticeable peaks appeared (Figure 2D), suggesting the presence of four different conformational states during the miRNA-target recognition as well as the exclusion of cleavage due to the existence of bulges caused by the central mismatches.19 The dynamics of miRNA-target recognition in the perfect match group (let-7a-3 and the target) was further investigated. As shown in Figure 3A,B, each miRNA binding event caused an instantaneous increase in both the donor and the acceptor fluorescence signals under the excitation of a 561 nm laser. In order to compare the annealing rate triggered by the spontaneous base paring with that modulated by the miRISC, the fluorescence trace was fit with the compatible exponential association function. As shown in Figure 3A, single exponential association function fit well to the curves of the acceptor in the absence of HeLa cell lysate and gave a half-time of 61.9 ± 9.0 ms, similar to that of the donor trace (68.5 ± 10.5 ms) (Figure 3C). However, in the presence of Hela cell lysate, the fluorescence time trace of the acceptor displayed a two-phase 5099

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Figure 3. “Two-state” formation of the miRNA-target duplex with perfect base pairing. (A and B) Top: fluorescence time traces of the donor (green line) and the acceptor (red line) obtained from the spontaneous annealing (A) and in the presence of HeLa cell lysate (B). Bottom: FRET analysis (blue line) of miRNA binding to a single target. (C) Comparison of the half -time of pairing in the absence (acceptor: red ■; donor: green ■) and in the presence (fast phase: white □; slow phase: black ■) of HeLa cell lysate. (D) Comparison of the FRET efficiency at plateau in the absence (white □) and in the presence (black ■) of HeLa cell lysate.

Figure 2. Histograms of FRET efficiency. (A and B) Histograms of FRET efficiency obtained from the perfect match group (let-7a-3 and the target) in the absence (A) and in the presence of HeLa cell lysate (B), respectively. (C and D) Histograms of FRET efficiency obtained from the seedless match group (miR-24 and the target) in the absence (C) and in the presence of HeLa cell lysate (D), respectively. Fits of individual subpopulations were shown as red lines for clarity.

association (Figure 3B). The miRNA first bound to the target with a short half-time of 64.7 ± 9.5 ms as spontaneous base pairing, and then, the annealing slowed down with a long halftime of 1.12 ± 0.27 s (Figure 3C). This two-phase binding suggested a “two-state” model for the miRNA-target recognition in real time.31,32 Considering that Ago adopted a compact conformation when containing guide DNA strand,20 the retardation in the duplex zippering induced by the miRISC suggested that the base pairing could not propagate without a substantial conformational transition in the miRISC. Moreover, the FRET efficiency at plateau in the presence of HeLa cell lysate (E = 0.43 ± 0.07) was higher than that in the absence of HeLa cell lysate (E = 0.36 ± 0.05) (Figure 3D), suggesting that the distance between the donor and the acceptor comparatively shrank due to the conformational change of the silencing duplex within the miRISC. The shrinkage of the silencing duplex may also explain the enhanced affinity between the guide and the target over the seed induced by “PIWI lobe” of Ago.33 The dynamics of miRISC-involved miRNA-target recognition in the seedless match group (miR-24 and the target) was investigated as well. The majority of miR-24 molecules (91%) which preincubated with HeLa cell lysate failed to form a miRNA-target duplex, displaying the same dynamic behavior as the miR-24 alone (see the Supporting Information, Figure S1). However, a few preincubated miRNA molecules (9%) were still observed to wound the target in discrete steps (Figure 4A,B). Figure 4B shows the variance of FRET efficiency with time for the molecule shown in Figure 4A. To quantify the stepping behavior, we used a variational Bayesian approach to model the FRET traces26 and further built a transition density plot.34 The

Figure 4. Stepwise recognition between the miRISC and the target with poor seed match. (A) Representative fluorescence time traces of the donor (green) and the acceptor (red) during miRISC winding on a single target. (B) Variance of FRET efficiency with time for the molecule shown in A. A step-finding algorithm was used to fit the FRET trace (orange line). (C) Transition density plot. (D) Comparison of the averaged dwell time.

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2010CB732600), the National Natural Science Foundation of China (Grant No. 21075129), the Knowledge Innovation Project of the Chinese Academy of Science (Grant No. KGCX2-YW-130), the Guangdong Innovation Research Team Fund for Low-cost Healthcare Technologies, the Natural Science Foundation of Shenzhen City (Grant Nos. JC201005270327A and JC201005270322A), the Fund for Shenzhen Engineering Laboratory of Single-molecule Detection and Instrument Development, and the Award for the Hundred Talent Program of the Chinese Academy of Science.

transition density plot represented the two-dimensional histogram for pairs of FRET values before (FRET enter) and after (FRET exit) each transition.34 As shown in Figure 4C, three well isolated peaks emerged in the transition density plot, suggesting the involvement of four-step pairing, which was in accordance with the result in Figure 2D where another four peaks emerged on the FRET distribution in the presence of HeLa cell lysate. Moreover, the averaged FRET values of each plateau in Figure 4C almost matched the peak values in Figure 2D (Table 1). Notably, three isolated peaks in Figure 4C

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Table 1. Comparison of FRET Values Obtained by Gaussian Distribution with Those Obtained by the Stepwise Model peak value of Gaussian distribution first second third fourth

0.27 0.49 0.64 0.78

± ± ± ±

0.12 0.05 0.05 0.01

(1) Fabian, M. R.; Sonenberg, N.; Filipowicz, W. Annu. Rev. Biochem. 2010, 79, 351−379. (2) Guo, H.; Ingolia, N. T.; Weissman, J. S.; Bartel, D. P. Nature 2010, 466, 835−840. (3) Friedman, R. C.; Farh, K. K.; Burge, C. B.; Bartel, D. P. Genome Res. 2009, 19, 92−105. (4) Latronico, M. V.; Condorelli, G. Nat. Rev. Cardiol. 2009, 6, 419− 429. (5) Fineberg, S. K.; Kosik, K. S.; Davidson, B. L. Neuron 2009, 64, 303−309. (6) Cho, W. C. Biochim. Biophys. Acta 2009, 1805, 209−217. (7) Rivas, F. V.; Tolia, N. H.; Song, J. J.; Aragon, J. P.; Liu, J.; Hannon, G. J.; Joshua-Tor, L. Nat. Struct. Mol. Biol. 2005, 12, 340− 349. (8) Chendrimada, T. P.; Gregory, R. I.; Kumaraswamy, E.; Norman, J.; Cooch, N.; Nishikura, K.; Shiekhattar, R. Nature 2005, 436, 740− 744. (9) Yoda, M.; Kawamata, T.; Paroo, Z.; Ye, X.; Iwasaki, S.; Liu, Q.; Tomari, Y. Nat. Struct. Mol. Biol. 2010, 17, 17−23. (10) Yao, B.; Li, S.; Jung, H. M.; Lian, S. L.; Abadal, G. X.; Han, F.; Fritzler, M. J.; Chan, E. K. L. Nucleic Acids Res. 2011, 39, 2534−2547. (11) Muddashetty, R.; Nalavadi, V.; Gross, C.; Yao, X.; Xing, L.; Laur, O.; Warren, S.; Bassell, G. Mol. Cell 2011, 42, 673−688. (12) Ye, X.; Huang, N.; Liu, Y.; Paroo, Z.; Huerta, C.; Li, P.; Chen, S.; Liu, Q.; Zhang, H. Nat. Struct. Mol. Biol. 2011, 18, 650−657. (13) Lai, E. C. Nat. Genet. 2002, 30, 363−364. (14) Brennecke, J.; Stark, A.; Russell, R. B.; Cohen, S. M. PLoS Biol. 2005, 3, e85. (15) Bartel, D. P. Cell 2009, 136, 215−233. (16) Shin, C.; Nam, J. W.; Farh, K. K.; Chiang, H. R.; Shkumatava, A.; Bartel, D. P. Mol. Cell 2010, 38, 789−802. (17) Brodersen, P.; Voinnet, O. Nat. Rev. Mol. Cell Biol. 2009, 10, 141−148. (18) Lal, A.; Navarro, F.; Maher, C. A.; Maliszewski, L. E.; Yan, N.; O’Day, E.; Chowdhury, D.; Dykxhoorn, D. M.; Tsai, P.; Hofmann, O.; Becker, K. G.; Gorospe, M.; Hide, W.; Lieberman, J. Mol. Cell 2009, 35, 610−625. (19) Wang, Y.; Juranek, S.; Li, H.; Sheng, G.; Tuschl, T.; Patel, D. J. Nature 2008, 456, 921−926. (20) Wang, Y.; Sheng, G.; Juranek, S.; Tuschl, T.; Patel, D. J. Nature 2008, 456, 209−213. (21) Wang, Y.; Juranek, S.; Li, H.; Sheng, G.; Wardle, G. S.; Tuschl, T.; Patel, D. J. Nature 2009, 461, 754−761. (22) Joo, C.; Balci, H.; Ishitsuka, Y.; Buranachai, C.; Ha, T. Annu. Rev. Biochem. 2008, 77, 51−76. (23) Tan, G. S.; Garchow, B. G.; Liu, X.; Yeung, J.; Morris, J. P. t.; Cuellar, T. L.; McManus, M. T.; Kiriakidou, M. Nucleic Acids Res. 2009, 37, 7533−7545. (24) Roy, R.; Hohng, S.; Ha, T. Nat. Methods 2008, 5, 507−516. (25) Liu, J.; Carmell, M. A.; Rivas, F. V.; Marsden, C. G.; Thomson, J. M.; Song, J. J.; Hammond, S. M.; Joshua-Tor, L. Science 2004, 305, 1437−1441. (26) Bronson, J. E.; Fei, J.; Hofman, J. M.; Gonzalez, R. L., Jr.; Wiggins, C. H. Biophys. J. 2009, 97, 3196−3205.

averaged value of each step 0.23 0.41 0.60 0.87

± ± ± ±

0.05 0.12 0.14 0.05

coincided with the existence of three obstacles (two G:U wobbles and one mismatch) in the partially complementary helix of miR-24-target between the donor and the acceptor (Scheme 1C), suggesting that the involvement of stepwise pairing resulted from the accommodation of poor seed matches by the miRISC. Further comparison of the dwell times in Figure 4D showed that the triggering of first transition was a rate-limiting step in the formation of duplex after the miRISC docking, which may require spatiotemporal coincidence with the structural plasticity of miRISC for the accommodation of the mismatches in the miRNA-target duplex.

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CONCLUSIONS In summary, we employ smFRET for the first time to monitor the conformational dynamics of the miRISC-involved target recognition in the whole cell lysate in real time. Our results reveal the two-state mechanism for human miRNA-target recognition with perfect base pairing, suggesting a conserved pattern as prokaryotic guide DNA-mediated recognition.21 Besides the general rule of miRNA-target recognition, our study brings new insights into the structural plasticity of human miRISC to conciliate the mismatches in seed region, which has an important implication for comprehensive understanding of the role of miRISC in the target recognition as well as predicting more diverse modes of target recognition in mammals to be found in the future.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary Figure S1 and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 755 86392211. Fax: +86 755 86392299. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program 973 (Grant Nos. 2011CB933600 and 5101

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