Dynamics of Gene Silencing in a Live Cell: Stochastic Resonance

Mar 5, 2014 - It is observed that repeated unbinding and rebinding of siRNA (to target mRNA) occur before gene silencing. 16 273 on-time periods ...
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Dynamics of Gene Silencing in a Live Cell: Stochastic Resonance Shyamtanu Chattoraj,†,§ Shekhar Saha,‡,§ Siddhartha Sankar Jana,*,‡ and Kankan Bhattacharyya*,† †

Department of Physical Chemistry and ‡Department of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India S Supporting Information *

ABSTRACT: Binding of a specific siRNA to the target mRNA in a live cell (human breast cancer cell, MCF-7) is studied by confocal microscopy. The specific siRNA (labeled with a fluorophore, alexa 488) exhibits much higher intensity of fluorescence in the bound state than in the free (unbound) state. It is observed that repeated unbinding and rebinding of siRNA (to target mRNA) occur before gene silencing. 16 273 on-time periods (residence or dwell time of siRNA in bound form) are detected. They follow a strikingly simple pattern. All of the on-time periods are odd-integral multiples of 5.5 ± 0.05 ms. This is ascribed to stochastic resonance.

SECTION: Biophysical Chemistry and Biomolecules

R

investigated oscillating behavior of actin regulatory protein signaling complex and calcium oscillation inside a cell.19 The concept of SR was first proposed to explain the periodic recurrence of ice ages.21 SR refers to maximization of a weak signal (or information transfer) in a nonlinear system at an optimal value of noise and is widely used in signal processing, communications, and control.22−38 The sensory system of living beings (e.g., the mechano-receptor signaling systems of crayfish,24 crickets,25 cat, and monkeys26) routinely detects a very weak signal in the presence of large noise. In a sensory neuron, an external noise is coupled to a weak periodic (coherent) signal, and this causes enhancement of the signal. SR in different systems has been analyzed by several theoretical methods. Ross and coworkers developed an exactly solvable theoretical model for single-molecule kinetics involving two states.20 Matsumoto and coworkers detected SR in cytochrome c redox network.28 Gong et al. studied the effect of external and internal noise on SR during reduction of NO on platinum surface.29 Jana and Bagchi considered SR to explain intermittent fluctuations of high-density and low-density forms in liquid water.31 Garai et al. studied stochastic protein synthesis (translation) in ribosomes.32,33 Longtin et al. analyzed the sensory signal transmitted to brain by neurons, in terms of SR in a general bistable system driven by a periodic force.26 In the present work, we have followed the analysis of Longtin et al. to show that SR occurs during silencing of a gene inside a live cell.

ecent advancement of single-molecule spectroscopy has made possible direct observation of gene expression through motion of transcription factors along DNA.1−5 Gene expression is also regulated by short interfering RNA (siRNA). siRNA causes sequence-specific degradation and translational inhibition of mRNA (i.e., gene silencing).6−14 siRNA, a short double-stranded RNA (dsRNA) consisting of ∼21−23 nucleotides, originates from either endogenous or exogenous double-stranded RNAs (dsRNA).10,11 Inside a cell, siRNA first binds to a protein assembly known as RNA-induced silencing complex (RISC), containing several proteins (e.g., dicer, TRBP, and Ago 2).6−14 Subsequently, the specific siRNA binds to the complementary nucleotides of the target mRNA and silences a particular target gene. Schwille and coworkers used fluorescence correlation spectroscopy (FCS) to investigate RISC in a live cell.7 Schneider et al. studied the siRNA delivery and mRNA knockdown in combination with bispecific antibody and lipid-based nanoparticles in a human breast cancer cell, MCF7.8 However, very little is known about the kinetics of siRNA− mRNA interaction. In this work, we employ single-molecule fluorescence spectroscopy to study the kinetics of genesilencing in a live cell. We demonstrate that binding of a specific siRNA to mRNA in a live cell (human breast cancer cell, MCF-7) exhibits stochastic resonance (SR). Many biological systems display semiperiodic oscillations.15−38 For an enzyme−substrate complex, Lu and coworkers attributed such oscillations to conformational transitions.15,16 Baldini et al. demonstrated that switching between neutral (N) and anion (A) forms of green fluorescent protein (GFP) results in such oscillations.17 Hynne and coworkers studied the effect of glucose and acetaldehyde on the glycolytic oscillation in yeast.18 Camilli and coworkers © 2014 American Chemical Society

Received: January 24, 2014 Accepted: March 5, 2014 Published: March 5, 2014 1012

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Figure 1. (A) Real-time PCR: decrease in % of MYH9 mRNA with time. (B) Western blot: Inhibition of NMHC II-A expression by siRNA in MCF7 Cells. Note that siRNA specific to NMHC II-A reduces the expression of NMHC II-A compared with nonspecific siRNA (upper panel lanes 3 and 4 vs lanes 1 and 2). (C) Quantification of Western blot: Value was obtained by considering nonspecific siRNA treated band intensity as 100%.

higher intensity (Figure 3C,D). It is evident that for all time windows both the intensity and fluctuation for specific siRNA (red) is much higher compared with the NS siRNA (black). The increase in fluorescence intensity is a signature of specific binding events.1 We call this a “bright” period. During the bright period, the system exhibits a series of high-intensity spikes (Figure 3 and S1, Supporting Information). No such bright period or high-intensity spikes are observed at any time for specific siRNA in culture medium. The NS siRNA does not display such high-intensity peak either in the culture medium or inside the cell (Figure 3A−D, lower panels). We now explain the experimentally determined time period (on-time) for which the specific siRNA remains bound to mRNA using the model of SR.21−38 SR may be viewed as the motion of a particle in a double-well potential (Figure 4A).22−27 In the presence of a periodic driving force, Q(ω), and a random noise, ξ(t), in the overdamped limit the Langevin equation may be written as26,27

For the present study, we have used a sequence-specific siRNA covalently labeled with a fluorophore alexa 488 against mRNA of MYH9 gene. As a control, we used an alexa-488labeled nonspecific (NS) siRNA. The NS and the specific siRNA contain the same number of nucleotides, but they differ in homology, that is, the sequence of nucleotides (details given in the Supporting Information). Because of the lack of complementary sequence homology, the NS siRNA cannot bind to the target mRNA and hence is incapable of gene silencing. The sequence-specific degradation of mRNA was studied by real-time PCR using primers specific for MYH9 mRNA. Figure 1A shows the time-dependent degradation of MYH9 mRNA. Note that the specific siRNA is able to degrade 88 ± 2% mRNA within 48 h. We also monitored the expression of MYH9 gene at the protein level (i.e., synthesis of the protein NMHC II-A). Figure 1B,C shows that the specific siRNA reduces synthesis of NMHC II-A when compared with a NS siRNA (upper panel lanes 3 and 4 vs lanes 1 and 2, Figure 1B). Figure 1C reveals that synthesis of NMHC II-A in specific siRNA-treated cells is reduced by 98 ± 0.15% relative to the nonspecific (NS) siRNA treated cells. Figure 2A,B shows the confocal microscopic images for specific and NS siRNA inside the cell, MCF-7. Note, the

ẋ = −

d U (x ) + ξ(t ) + Q (ω) dx

(1)

In the bistable system described by Figure 4A, the “weak” periodic signal Q(ω) periodically lowers or raises the two wells (A and B) relative to the barrier.22−27 The “weak” signal, by itself, cannot excite the particle above the barrier to cause transition from one well to the other. On the contrary, the random noise ξ(t) is capable of inducing interwell hopping, albeit in a random fashion. The nonlinear coupling between the periodic signal and random noise makes the interwell transitions surprisingly regular, and the regularity improves with increase in noise up to an optimal level.23−38 The signalto-noise ratio in a system undergoing SR has been calculated for many different forms of U(x), driving force Q(ω), and random noise.27,37 We now elaborate the analogy of our system (siRNA) with the general model of SR described by Figure 4A. In our case, state A corresponds to specific siRNA in the unbound state (or in the RISC) in the cell. State B denotes specific siRNA bound to the target mRNA in the cell. The fluorescence intensity in state B is higher than that in state A. Thus the observed output is the increase in fluorescence intensity (from state B). The transition from A to B refers to specific binding of siRNA to mRNA, that is, reading the complementary nucleotide sequence in mRNA. Thus the coordinate x denotes binding of specific siRNA to mRNA and hence is described by the combined distance of the ∼22 nucleotides of siRNA from the complementary nucleotides in mRNA. Note that binding of

Figure 2. Microscopic FLIM images of control and specific siRNA in a live cancer cell, MCF-7: (A) Specific siRNA. (B) Nonspecific (control) siRNA.

specific siRNA binds specifically to its target mRNA, giving rise to high fluorescence intensity. Thus, the specific siRNA is visible in many locations spread over a very large region in the cell (Figure 2A). In contrast, the NS siRNA, which cannot bind to mRNA, is localized in a small region in the cell (Figure 2B). According to the single-molecule fluorescence fluctuations (Figure 3A), the specific siRNA exhibits a long initial “dark” period (low intensity) for ∼70 s. Beyond ∼70 s, when the siRNA binds to the specific site in the mRNA, it exhibits much 1013

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Figure 3. (A) Single-molecule fluorescence intensity versus time trace of specific (red) and nonspecific (black) siRNA in a live MCF-7 cell. (B−D) The same at different time windows.

Figure 4. (A) Stochastic resonance and transition between unbound (A) and bound (B) form of specific siRNA inside the cell. (B) On-time distribution (in state B) for specific siRNA in a live cancer cell, MCF-7. (C) On-time histogram for a specific siRNA in a live MCF-7 cell with a period T0/2 = 5.5 ± 0.05 ms.

specific siRNA bound to the mRNA (i.e., inverse of unbinding of ∼22 nucleotides of siRNA from mRNA). Similar (0.3−5 ms) residence times were previously reported for a transcription factor diffusing along DNA.2 Similar SR in gene expression has been previously reported in switching of phenotype of E. coli cells,3 in infection of E. coli by λ phage DNA,37 and in synthetic gene network.38 In our case, specific binding of siRNA to mRNA and consequent gene silencing involve a library of signaling molecules present in RISC. In this case, the random noise ξ(t) refers to fluctuation of basal concentration of signaling agents along with random polymer chain dynamics (Rouse Chain dynamics) of mRNA and siRNA. The periodic driving force, Q(ω) = Q0 sin ωt (i.e., the biological switch) may involve both structural fluctuation and oscillation of concentration (availability) of key signaling agents. An example of structural fluctuations includes dissociation of repressors from DNA, giving rise to sudden bursts in gene expression,3 and intermittent coherence in conformational transitions of an enzyme−substrate complex.15,16 In our case, the structural fluctuation may be the cooperative motion of mRNA, siRNA, and signaling molecules along with the hydrogen bonds between the complementary nucleotides. The oscillation of concentration of chemical inducers is implicated in infection of E. coli by λ phage DNA.37 Most recently, many microfluidic devices have been developed that perturb synthetic gene circuits by controlled addition of chemical inducers.38 The exact nature and function of the signaling molecules involved in binding of siRNA and mRNA is still unknown and is an interesting unsolved problem.

siRNA to mRNA does not immediately cause silencing of the gene (degradation and translational inhibition of mRNA). The fluctuations in fluorescence intensity (e.g., Figure 3) suggest that many cycles of binding (hopping from A to B) or unbinding (B to A) occur before silencing. The binding−unbinding represents the ABBA sequence proposed by Longtin et al.26 For a sinusoidal driving force, Q(ω) = Q0 sin ωt, in the first half-period, T0/2 (ωt = 0 − π), the sign of the periodic force Q (= Q0 sin ωt) (Figure 4A) is positive, denoting A-to-B (AB) transitions. For the second halfperiod (ωt = π − 2π), Q is negative, which corresponds to B-toA (BA) transitions.26 The probability of AB transitions is maximum at ωt = π/2. After this, the reset BA transition occurs with maximum probability at ωt = 3π/2, and hence the system stays in state B for a period T0/2 (between ωt = π/2 and 3π/2). If, by chance, the BA transition does not occur at ωt = 3π/2, the system would remain in state B until ωt = 3π/2 + 2π= 3T0/ 2. Thus, the time period over which the system stays in state B (“on-time,” or “dwell-time” Figure 4B) is an odd-integral multiple of T0/2.26 In our case, the on-time is determined experimentally from the period of high intensity (“bright period”, Figure 3C,D). Figure 4C shows the distribution of the on-time periods (in total 16 273 periods were detected) for specific siRNA bound to mRNA in the cell. The inverse of ontime gives rate constant of unbinding of siRNA (escape from well B). The on-time distribution (Figure 4C) exhibits remarkable similarity to the on-time histogram previously reported for the ABBA sequence.26 It is readily seen that the modes of the on-time distribution are odd-integral multiples of a period T0/2 = 5.5 ± 0.05 ms. The observed on-time of 5.5 ± 0.05 ms may be interpreted as the residence (dwell) time of the 1014

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Notes

Figure 5 shows distribution of high-intensity spikes (for specific siRNA) with time. It is readily seen that there is an

The authors declare no competing financial interests.

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ACKNOWLEDGMENTS We thank the Department of Science and Technology, India (Centre for Ultrafast Spectroscopy and Microscopy Project and J. C. Bose Fellowship) and the Council of Scientific and Industrial Research (CSIR) for generous research support. S.C. thanks CSIR and S.S. thanks DST for awarding fellowships.

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Figure 5. Distribution of high fluorescence intensity spikes of specific siRNA in a live MCF-7 cell at various time windows.

initial dark period (0−70 s). During the initial “dark” period, the specific siRNA “searches” for the specific site of the mRNA. Similar “searching time” of ∼60 s (1 min) has been reported for searching by a repressor for a specific sequence in DNA.2,3 It is readily seen that the high-intensity spikes gradually disappear after ∼160 ± 5 s, indicating silencing of the gene. Thus degradation of mRNA or gene silencing occurs on the ∼160 s time scale. In summary, this study demonstrates that in a live cell, binding of a specific siRNA to mRNA involves an initial search (dark period), followed by repeated unbinding and rebinding processes. Second, the transitions from bound to unbound form of siRNA involves odd integral multiple of 5.5 ms, which is in good agreement with the model26 of a bistable system, modulated with a periodic signal. Using single-molecule fluorescence, Xie and coworkers demonstrated that gene expression is a stochastic single-molecule event.3 Wang and coworkers have investigated the existence of such oscillation and bistability due to post-transcriptional mRNA degradation or translational inhibition using computer simulation.13 They have also shown stochasticity associated with gene regulation.13 In our case, the periodic signal is inherent in the system and induces transition (switching) between the two states (bound and unbound form) of the specific siRNA inside the cell. The periodic signal is associated with binding of siRNA to mRNA. Further investigation on the origin of this intrinsic periodic signal may provide a deeper insight into the mechanism of siRNA-mediated gene silencing. This may have potential interest for gene expression and its regulation for therapeutic purpose. The generality of this phenomenon in other types of cells will be discussed in future work.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental procedure and materials and methods. Comparison of intensity versus time trace of specific and nonspecific (control) siRNA in different time windows. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (S.S.J.). *E-mail: [email protected] (K.B.). Author Contributions §

S.C. and S.S. contributed equally. 1015

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