A Dynamic Search Process Underlies MicroRNA Targeting

Abstract

Argonaute proteins play a central role in mediating post-transcriptional gene regulation by microRNAs (miRNAs). Argonautes use the nucleotide sequences in miRNAs as guides for identifying target messenger RNAs for repression. Here, we used single-molecule FRET to directly visualize how human Argonaute-2 (Ago2) searches for and identifies target sites in RNAs complementary to its miRNA guide. Our results suggest that Ago2 initially scans for target sites with complementarity to nucleotides 2-4 of the miRNA. This initial transient interaction propagates into a stable association when target complementarity extends to nucleotides 2-8. This stepwise recognition process is coupled to lateral diffusion of Ago2 along the target RNA, which promotes the target search by enhancing the retention of Ago2 on the RNA. The combined results reveal the mechanisms that Argonaute likely uses to efficiently identify miRNA target sites within the vast and dynamic agglomeration of RNA molecules in the living cell.

Figure 1. Single-Molecule Observation of Ago2-miRNA Target Recognition

(A) Illustrated schematic representation of our single-molecule FRET assay. (B) Sequences of miRNA and a target RNA with N (the length of the matched region between miRNA and target RNA) equal to 6 nt. The donor fluorophore (Cy3) is positioned on the 9th nt of miRNA (counting from the 5′ end of miRNA) and the acceptor (Cy5) on target RNA opposite nt 17 of miRNA. Vertical lines denote contiguous base pairs between the guide and target. A dot “.” represents an inconsecutive pair. (C) CCD images (donor channel) show Ago2-miRNA binding to target RNA. Individual spots represent single-molecule complexes. (Left) Result representative of experiments with N = 6. (Middle) The same as left but without Ago2 included. (Right) U₆₃ used as a negative control while Ago2 was included. Scale bar 5 µm. (D) The number of accumulated binding events plotted as a function of time for the three cases in [C]. (E) A fluorescence time trace obtained (with a resolution 100 msec) shows three events of docking and dissociation of Ago2-miRNA at a single spot in the microfluidic chamber. Black arrows indicate binding of Ago2-miRNA to a target mRNA. Gray arrows indicate dissociation. (F) A FRET histogram obtained from 125 single-molecule traces fitted with a Gaussian function. (G) The dwell time of binding (Δτ) was fitted with a single exponential decay. The first column of the data was not included in the analysis to avoid potential artefacts arising from the limit of the time resolution. Error is the standard deviation (std) of 3 independent experiments that were carried out on different days. The bin size is 1 sec. See also Figure S1.

Figure 2. Kinetics of MicroRNA Target Recognition

(A) Sequences of miRNA and a target RNA with various values of N. Vertical lines represent consecutive base paring between the guide and the target RNAs. Colons “:” represent potential GU wobble pairs. Dots “.” represent inconsecutive pairs. “p” represents 5′ phosphate in the guide miRNAs. The biotin at 3′ end of target RNAs is denoted by “b”. The sequence of the miRNA with N = 19 is hsa-let-7a. The sequences of other miRNAs were derived from hsa-let-7a with variations at 5′ and 3′ ends. The sequences of the miRNA with N = 3 and 4 contain A instead of genomic sequence U at nt 6. The sequence of the miRNA with N = 7 contains A instead of genomic sequence U at nt 9 and has a dye conjugated at nt 10. (B) Representative time traces for the interaction between target RNAs and Ago2-miRNA complexes with different values of N. A time resolution of 100 msec was used for the negative control and N = 3 ^{(nt 5-7)}, 3 ^{(nt 2-4)}, 4, 5, and 6. A time resolution of 300 msec was used for N = 7, 8, 15, and 19. (C) Dwell time histograms for different values of N. The dwell time (Δτ) of N = 3 ^{(nt 5-7)}, 3 ^{(nt 2-4)}, 4, 5 and 6 is 0.2 ± 0.1, 0.7 ± 0.2, 0.8 ± 0.1, 1.2 ± 0.3, and 1.9 ± 0.1 sec, respectively. Error is the std of 3 independent experiments carried out on different days. The bin size for N = 3 ^{(nt 5-7)} is 0.5 sec. The bin size for N = 3 ^{(nt 2-4)}, 4, 5 and 6 is 1 sec. The dwell time of N = 7, 8, 15, and 19 was not determined (N. D.) due to photobleaching. The dwell time of the negative control is not available (N.A.) due to a lack of binding events. (D) A bar plot shows the dependence of dwell time on N. The dashed red line indicates the observation time limit (300 sec), which is constrained by photobleaching. Error bars are the std of 3 independent experiments that were carried out on different days. (E) Binding rate plotted for different values of N. In all the cases, 1 nM Ago2-miRNA was introduced to the chamber. The error bars are the std of 3 independent experiments from different days. See also Figure S2 and Table S1.

Figure 3. Lateral Diffusion of Ago2-miRNA

(A) Illustrated is the schematic representation of the single-molecule FRET assay used to observe lateral diffusion of Ago2-miRNA between tandem binding sites before dissociation occurs. N₁ and N₂ are the lengths of matched sequence between the miRNA and sites 1 and 2, respectively. (B) Sequences of N₁ = N₂ = 6 target sites paired to guide miRNA. The donor (Cy3) is positioned at the 9th nt of miRNA. The acceptor (Cy5) is positioned in site 1, opposite nt 17 of the paired miRNA. When miRNA is bound to site 1, the two dyes are separate by 7 nt, leading to high FRET (E ~ 0.75). When it is bound to site 2, they are separate by 13 nt, leading to low FRET (E ~ 0.5). (C) Fluorescence signals in a time trace obtained with a time resolution 100 msec reports on docking and dissociation of Ago2-miRNA. Intensity of FRET signal indicates position of Ago2-miRNA complex with respect to sites 1 and 2 (site 1, high FRET; site 2, low FRET). (D) (Top left) A FRET histogram was fit with two Gaussian functions (E ~ 0.5 and 0.75). Dwell time distribution of total binding (Δτ , top right), binding to site 2 (Δτ₂, bottom left), and binding to site 1 (Δτ₁, bottom right). The distributions were fit with a single exponential decay. The first column of the data was not included in the analysis to avoid artefacts from the limited time resolution. Error is the std of 3 independent experiments that were carried out on different days. The bin size is 1 sec. (E) A bar graph shows the dependence of the dwell times (Δτ₁ and Δτ₂) on the length of the matched region between miRNA and each binding site. Error is the std of 3 independent experiments that were carried out on different days. See also Figure S3 and Table S1.

Figure 4. Three-color FRET Assay Detecting Lateral Diffusion

(A) Illustrated is the schematic representation of the single-molecule three-color FRET assay used to observe lateral diffusion of Ago2-miRNA between tandem binding sites. N₁ and N₂ are the lengths of matched sequence between the miRNA and sites 1 and 2, respectively. An equal amount of Cy3 (green, donor 1) and Cy5 (red, donor 2) labeled miRNA is added into a chamber which has a Cy7 (blue, acceptor) labeled tandem target construct immobilized. (B) Sequences of N₁ = N₂ = 6 target sites paired to guide miRNA. The donor (Cy3 or Cy5) is positioned at the 9th nt of miRNA. The acceptor (Cy7) is positioned in site 1, opposite nt 17 of the paired miRNA. (C) Fluorescence signals in time traces were obtained with a time resolution 500 msec. (Left) A time trace showing alternations in Cy3-Cy7 FRET efficiency. (Right) A time trace showing alternations in Cy5-Cy7 FRET efficiency. The horizontal lines in FRET efficiency plots indicate two different FRET states.

Figure 5. A synergistic effect from two neighboring target sites

(A) A fluorescence time trace obtained using a single target construct (N = 6) in the presence of 10% PEG with a time resolution 300 msec. (B) A dwell time histogram for the condition used in [A]. The dwell time (Δτ) was 10.5 ± 2.7 sec. Error is the std of 3 independent experiments that were carried out on different days. The bin size is 20 sec. (C) A fluorescence time trace obtained using a tandem target construct (N₁ = N₂ = 6) in the presence of 10% PEG with a time resolution 300 msec. (D) A dwell time histogram for the condition used in [C] was fitted using a double exponential curve (green). The double exponential fit resulted in two characteristic times Δτ₁ (61%), 13.9 ± 1.7 s, a blue curve; Δτ₂ (39%) = 76.0 ± 22.7 s, a yellow curve). The average, <Δτ> = 31.4 ± 8.4 sec, was estimated from Δτ₁ × 61% + Δτ₂ × 39%. A peak at Δτ > 900 (sec) is due to a limited observation time and was not included during the fitting. Error is the std of 3 independent experiments that were carried out on different days. The bin size is 20 sec. (E) The total dwell time plotted for different concentrations (weight to volume) of PEG. Error is the std of 3 independent experiments. See also Figure S5.

Figure 6. Role of the PAZ Domain in Lateral Diffusion

(A) A time trace obtained with a time resolution 300 msec shows lateral diffusion that occurs between two binding sites (N₁, high FRET; N₂, low FRET, each 15 nt). (B) A FRET histogram was obtained under the condition of [A] and was fit with Gaussian functions (E ~ 0.5 and 0.75). (C) A time trace was obtained with a time resolution 300 msec using a miRNA construct that was modified at its 3′ end with biotin. (N₁, high FRET; N₂, low FRET, each 15 nt). (D) A FRET histogram was obtained under the condition of [C] and was fitted with Gaussian functions (E ~ 0.5 and 0.75). (E) The kinetic rate of shuttling between two binding sites (N₁ and N₂) was measured with N_i = 6, 8, “7+10”, 15, and 19 using unmodified and 3′ biotinylated miRNA constructs. Error is the std of 3 independent experiments that were carried out on different days. See also Figure S4.

Figure 7. A model for target search and recognition by human Ago2-miRNA

(A) Illustration of proposed steps in target recognition by Ago2-miRNA. Ago2-miRNA binds the single stranded target RNA and diffuses along the RNA rapidly—sliding over 69 nt (the length of our target construct) in less than 100 msec (our time resolution). Complementary sites of N ≥ 3 cause the complex to pause and remain meta-stably bound (on the order of 1 second). When site complementarity extends the full length of the seed (nt 2–8) or longer, the complex remains bound stably for extended times (>>300 sec). See also Figure S6.