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Argonaute Facilitates the Lateral Diffusion of Guide along Its Target and Prevents Guide from Being Pushed Away by Ribosome Guangtao Song, Hui Chen, Gang Sheng, Yanli Wang, and Jizhong Lou Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00213 • Publication Date (Web): 28 Mar 2018 Downloaded from http://pubs.acs.org on March 29, 2018
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Biochemistry
Argonaute Facilitates the Lateral Diffusion of Guide along Its Target and Prevents Guide from Being Pushed Away by Ribosome Guangtao Song,†,⊥ Hui Chen,†,‡,⊥ Gang Sheng,† Yanli Wang,*†,‡ and Jizhong Lou*†,‡ †
Key Laboratory of RNA Biology, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
‡
University of Chinese Academy of Sciences, Beijing, 100084, China
Supporting Information ABSTRACT: Argonaute (AGO) proteins play central roles in nucleic acid-guided interferences that regulate gene expression and defend against foreign genetic elements in all life. Although much progress has been made on the function of argonaute proteins in target recognition and cleavage, the detailed mechanism of their biological functions is not fully under-stood. Here, using AFM-based single-molecule force spectroscopy, we studied the target-guide dissociation in the absence or presence of Thermus thermophiles AGO (TtAGO). Our results indicated that AGO changed the fundamental properties of target-guide interaction. Target dissociates easier from guide in the lateral direction of nucleic acid in the presence of AGO protein, but harder in the longitudinal direction. Our results support the idea that the onedimensional diffusion of RISC along target strand is more efficient than that of three-dimensional diffusion and explain the priority of RISC binding over ribosome complex during translation elongation.
MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) are small regulatory RNAs broadly involved in many levels of genome function, including chromatin structure, chromosome segregation, transcription, RNA processing, RNA stability, and translation.1-6They exert their regulatory functions through the formation of ribonucleoprotein complexes termed RNA-induced silencing complexes (RISCs).5-7Argonaute (AGO), a highly conserved protein from prokaryotes to eukaryotes, forms the functional core of RISCs. It provides anchor sites for the microRNA (miRNA) guide strand and uses the sequence information encoded within the loaded guide to identify target mRNAs through Watson-Crick base-pairing for regulation.8-14 AGO proteins are composed of four domains: N-terminal (N), middle (Mid), P-element Induced WImpy testis (PIWI) and PiwiArgonaute-Zwille (PAZ) domains.11, 14, 15 The overall protein structure is bi-lobed, with one lobe consisting of the PAZ domain and the other lobe consisting of the PIWI domain flanked by N and Mid domains (Supplemental Figure S1A). Structural and biochemical studies revealed that AGO protein divides the loaded guide strand into five domains with distinct functions: 5’ anchor (g1), seed (g2-g8), central (g9-g12), 3’ complementary (g13-g16), and 3’ tail region (g17-g21) (Supplemental Figure S1B). The 5’ anchor and 3’ tail region of guide RNA are anchored within Mid
and PAZ pockets, respectively.16, 17 The seed sequence initiates target binding and is the primary determinant of binding specificity.18 The central region needs to base pair with target in order to initiate the target cleavage catalyzed by the PIWI domain which adopts an RNase H-like fold.19 The 3’ supplementary region is thought to complement seed pairing for some miRNA targets. In addition to remarkable advances in our understanding of the mechanism of target recognition and endonucleolytic cleavage by RISC, the kinetic aspects of this process have been studied extensively, especially by the use of single-molecule fluorescence approaches.18, 20-26 Structures of RISCs indicated that seed region of guide strand is arranged in an A-form-like helical geometry by AGO, which reduces the entropic cost inherent to base pairing and make productive collisions with targets more likely.8-10, 13, 27, 28 Single-molecule studies further revealed that AGO proteins accelerate target-guide association by up to 250-fold, with increasing importance toward the 5’-end of the guide.22, 24, 25 Surprisingly, the target release after cleavage also occurs faster than the individual duplexes, and release of either cleavage products accelerates removal of the second, indicating that AGO generates an environment that actively promotes the release of the cleavage products.24, 25 In addition, Chandradoss et al. showed that neighboring sites on the same miRNA resulted in higher residence times of RISC on the target than expected, they proposed that transient sub-seed interactions might enable lateral diffusion of RISC along RNA molecules, thereby temporarily reducing in the search space from three to one dimension.22 All these results indicated that AGO generates an environment that actively promotes the release of the target strand, especially at the lateral direction of target strand. The molecular mechanism of the distinct role of AGO in target association and dissociation is still not completely clear. To address these questions, by using TtAGO as the model system, we studied the effect of AGO on the guide-target dissociation process by atomic force microscopy (AFM) based singlemolecule force spectroscopy. We first measured the unbinding force between guide DNA and target strand with AFM by pulling at the adjacent 3’-5’-ends (unzipping mode, Figure 1) or the opposite 3’-ends (stretching mode, Figure 2). The target oligonucleotide with a 5’-SH (for unzipping mode, Figure 1A) or 3’-SH (for stretching mode, Figure 2A) modification was immobilized via Au-S interaction on gold coated
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AFM cantilever. A (dT)16 spacer sequence was used to eliminate the surface effect. Let-7a guide strand with 5’-phosphorylated and 21th biotin modified dT was immobilized via biotin-streptavidin interaction on the biotin-PEG-functionalized glass surface. During the experiment, a target DNA-coated AFM cantilever was brought into contact with the guide DNA immobilized on the glass surface, and then the guide-target duplex could form (Figure 1 and 2). When the AFM tip is separated from the surface, the dsDNA can be broken and the unbinding force can be recorded. The most probable unbinding force can be determined by Gaussian fits to those force distribution histograms.
Figure 1. Effect of AGO on target dissociation at unzipping mode. (A), (C) Schematic of the experimental setup in the absence (C) or presence (A) of AGO proteins. (B),(D) The probability distribution of the unbinding forces of the guide-target duplex (B) or RISC-target complex (D). Inlet in (B), a typical forceversus-piezo displacement for the DNA duplex during the retraction of the sample with a velocity of 200 nm/s.
Figure 2. Effect of AGO on target dissociation at stretching mode. (A), (C) Schematic of the experimental setup in the absence (A) or presence (C) of AGO. (B),(D) The probability distribution of the unbinding forces of the guide-target duplex (B) or
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RISC-target complex (D). Inlet in (B), a typical force-versuspiezo displacement for the DNA duplex during the retraction of the sample with a velocity of 200 nm/s. It has been well defined that, when pulling dsDNA along with the stretching mode, a force induced transition between B-DNA and highly overstretched S-DNA occurs at a force of 65 pN will be detected.29-32 For short DNA oligonucleotides, the unbinding between complementary DNA strands mainly occurs during the B-S transition, i.e., around 65 pN.33, 34 In contrast, when pulling a dsDNA with the unzipping mode, Strand separation occurred abruptly at 12–13 pN and displayed a reproducible ‘saw-tooth’ force variation pattern with an amplitude of +/– 0.5 pN along the DNA, which is much lower than that of stretching mode.32, 35, 36 Therefore, the dissociation between complementary DNA strands is geometry dependent, where the lateral separation alone the DNA is much harder than that of vertical/longitudinal direction. As expected, the rupture force histogram between the DNA strands is very different between the unzipping mode (Figure 1B) and stretching mode (Figure 2B), both can be well fitted by single Gaussian distribution (Figures 1B and 2B, blue curves), results in optimal rupture force 21.7pN for unzipping and 61.3 pN for stretching (Table 1). These optimal rupture forces close to those of previous studies,32, 35, 36 confirming the validity of our approaches. We next studied the effect of AGO on the unbinding between guide and target strands. In these experiments, the guide DNA is pre-incubated with AGO protein for 30 min before immobilized on the surface (Figure 1C and Figure 2C). As seen in Figures 1D and 2D, in the presence of AGO, the rupture force histogram changed significantly and cannot been fitted well with single Gaussian, whereas double Gaussian fits can generate better results (Figures 1D and 2D, blue curves). In the unzipping mode, in addition to the peak correlated to that in the absence of AGO (Figure 1D, red curve), a new much higher force peak around 79 pN appeared (Figure 1D, green curve), indicates that AGO increase the energy barrier for the interaction between guide DNA and its target along this longitudinal direction. However, in the stretching mode, other than the peak correlates with that in the absence of AGO (Figure 2D, green curve), a new much lower force peak around 20 pN appeared (Figure 2D, red curve), indicates that the separation between guide DNA and target strand alone the lateral direction is much easier when adding AGO into the system, with the dissociation energy barrier is much lower. All these data showed that AGO reshape the fundamental properties for target dissociation with guide which has been found in other experiments.25 In some organisms, the guide strand usually matched perfectly and bound stably with the target. After this stable binding, RISC selectively cleaves the target strand at the position facing nucleotides 10 and 11 of the guide, and leads to the release of cleavage products. But in some other organisms, especially in animals, the guide strands often don’t match the target very well, which do not facilitate target cleavage and the interferences take other pathways. Moreover, previous single-molecule fluorescence studies showed that, in contrast to the directionality of small RNA binding, which always occur from 5’- to 3’-, the release of the products or the dissociation of target-guide duplex follows no strict order but largely depends on the relative base-pairing stability of the fragments.26 These results are not quite in consistent with the fact that AGO protein divides the guide strand into five domains with distinct functions. To address these questions and further study the directional preference of product release/target guide dissociation, we next used two different targets with 5’- or 3’- mismatched sequences. The dissociation histograms are shown in Figure 3 and 4 respectively, and the single or double Gaussian fits parameters
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Biochemistry are listed in Table 1. When using target DNA with nt 11-21 mismatched sequence (seed pairing, Figure 3), the unzipping force increases (Figure 3A and 3C), while the stretching force decreases (Figure 3B and 3D), in the presence of AGO. This is quite in agreement with that of WT target. However, when using the target DNA with 1-10 mismatched (tail pairing, Figure 4), the unzipping force increases (Figure 4A and 4C), while the stretching force does not differ significantly in the presence of AGO (Figure 4B and 4D). These results revealed that the dissociation with seed pairing target is easier than that of tail pairing target, indicating that AGO protein play different roles in remodeling different region of the guide strand. Comparing with wild-type target, it could also be found that target dissociation behavior of RISC is mainly determined by the seed region of guide DNA. It is known that the seed region of guide DNA is constrained in an A-form geometry by AGO in RISC. As the results, when using the stretching mode to dissociate guide strand from its target, the stretching B-S transition will be destroyed by AGO, leading to a decrease of rupture force. In contrast, when using the unzipping mode, guide DNA along with AGO in RISC serves as a DNA binding protein. And as expected, the unbinding force is similar to that of single-stranded DNA with DNA binding proteins (around 50 pN).
dimensional diffusion of RISC along target strands is more efficient than that of three-dimensional diffusion.37 That is, argonaute facilitates target search by assisting the lateral diffusion of guide strand along its mRNA target.
Figure 4. Effect of AGO on tail-pairing target dissociation. (A), (C) The probability distribution of the unbinding forces of the guide-tail-pairing target duplex (A) or RISC-target complex (C) at the unzipping mode. (B), (D) The probability distribution of the unbinding forces of the guide-seed pairing target duplex (B) or RISC-target complex (D) at the stretching mode. Table 1.AGO effect on unbinding force of guide-target duplex calculated from Gaussian fitting of the probability distribution of the unbinding forces in Figures 1-4.
Figure 3. Effect of AGO on seed-pairing target dissociation. (A), (C) The probability distribution of the unbinding forces of the guide-seed pairing target duplex (A) or RISC-target complex (C) at the unzipping mode.(B), (D) The probability distribution of the unbinding forces of the guide-seed pairing target duplex (B) or RISC-target complex (D) at the stretching mode. In finding its optimal target position, Ago2 scans the target sequences via initial three nucleotides match at position 2-4 of the miRNA. It is suggested that lateral diffusion play important roles in this process. While to diffuse laterally, the RISC complex needs to break with the initial target and binds again to the next target site. To ensure lateral diffusion, the breaking of the initial target then uses a way similarly as the stretching mode in our studies (especially 1D sliding, Supplemental Figure S2A). Our results showed that the presence of TtAGO reduces the energy barrier and dissociates the guide and target duplex at lower forces, which indicates that the RISC release on the lateral direction of target strand is much easier than guide-target hybrid alone. Thus, our results provide additional supports to the finding that the one-
Previous studies showed that when bound to the open reading frame, plant AGO1-RISC can block the movement of ribosome, which suggested that the force for the binding between RISC and mRNA target is stronger than that for the translational elongation.38 When ribosome moves along mRNA and encounters the target-bound RISC, the elongation force tends to dissociate the RISC/target interaction in an unzipping manner (Supplemental Figure S2B). It has been shown that the translation can still happen at force up to 15 pN, which is compatible with that of the unzipping of DNA duplex in the absence of AGO protein.39 As shown in our studies, the guide/target unzipping force increased (>40pN) when TtAGO is present and RISC is formed, which suggests that AGO protein helps to stabilize guide/target duplex when competing with ribosome, prevents the guide strand from being pushed away by ribosome, terminates protein translation, thus results in translational repression. Of course, this effect can also be enhanced by recruiting other molecules such as GW182 to the target binding site.
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In summary, by using AFM based single-molecule force spectroscopy, we studied the strand dissociation of target DNA from RISC at different directions. Our results show the presence of TtAGO increase the energy barrier for dissociation from lateral direction (unzipping), and decrease the energy barrier for dissociation for longitudinal direction (stretching). Our results provide additional supports for the one-dimensional diffusion model for RISC to find its target and explains the priority of RISC for mRNA when compete with ribosome. Our results also suggest the dissociation pathway for RISC and target strand (or cleavage product), i.e. the pathway along lateral direction should be preferred.
ASSOCIATED CONTENT Supporting Information Supporting Methods and Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author Yanli Wang,
[email protected] Jizhong Lou,
[email protected].
Author Contributions ⊥
These authors contributed equally.
Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT This work was supported by the National Basic Research Program of China (2014CB910202 to Jizhong Lou) and National Natural Science Foundation of China (91219103 to Jizhong Lou, 31300772 to Guangtao Song, and 31222022 to Jizhong Lou).
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