RNA Hairpin Folding in the Crowded Cell

Abstract Precise secondary and tertiary structure formation is critically important for the cellular functionality of ribonucleic acids (RNAs). RNA folding studies were mainly conducted in vitro, without the possibility of validating these experiments inside cells. Here, we directly resolve the folding stability of a hairpin‐structured RNA inside live mammalian cells. We find that the stability inside the cell is comparable to that in dilute physiological buffer. On the contrary, the addition of in vitro artificial crowding agents, with the exception of high‐molecular‐weight PEG, leads to a destabilization of the hairpin structure through surface interactions and reduction in water activity. We further show that RNA stability is highly variable within cell populations as well as within subcellular regions of the cytosol and nucleus. We conclude that inside cells the RNA is subject to (localized) stabilizing and destabilizing effects that lead to an on average only marginal modulation compared to diluted buffer.

labeled 4U RNA dissolved in ddH 2 O were loaded to a Femtotips II glass-capillary (Eppendorf) from the back using microloader capillaries (Eppendorf). Injection pressure was adjusted to 100-250 hPa for 0.2-0.7 s so that no visible expansion of cell volume was observed during injection.
Sample preparation for FReI. Stock solution of FRET-labeled 4U RNA sample was heated up to 70°C for 5 min and slowly cooled down to RT to ensure correct folding of the RNA hairpin structure. For in vitro measurements, 5 µM samples were prepared by mixing the RNA with different crowding solutions. 20 µL of each mixture were placed between a glass bottom dish (FluoroDish, WPI) and a glass cover slip using a 100 µm imaging spacer (Sigma-Aldrich). The dish was then placed on the microscope stage and the excitation light was focused onto the imaging spacer which represents the solution layer. Comparative in-cell measurements were performed in the same way. After removing the medium a glass cover slip with 80 µL Leibovitz's L15 medium supplemented with 30% FBS was mounted via an imaging spacer onto the glass bottom dish which contained microinjected cells. The RNA stock solution was used directly as microinjection sample.
FReI measurement. The principles of Fast Relaxation Imaging (FReI) were described earlier. [3][4][5] In this study, an inverted fluorescence microscope (AxioObserver Z1, Zeiss) with a Colibri excitations system was used. The monochromatic light was guided through a 90° reflection using a beamsplitter (DFT 490+575, Zeiss) to a 40x objective (N.A. = 0.95, Zeiss) and focused on the sample. Emitted fluorescence of Atto488 and Atto565 was transmitted through the first beamsplitter and was reflected by a prism to the second beamsplitter (BC 565, Zeiss), where donor and acceptor/FRET fluorescence light were separated and reflected onto different CCD cameras (AxioCam HS, Zeiss) with appropriate emission filters (BP512/30 and BP630/98, Zeiss). As described previously, 5 a diode laser (m2k laser; λ = 2200 nm) was used to induce fast 2.4 °C-temperature jumps, heating the cells and the entire field of view (40x objective; N.A. = 0.95) homogenously. For each jump the temperature was equilibrated for 20 s. The temperature jumps were calibrated using the temperature-sensitive dye rhodamine B. 5 During temperature jump stepping, the power profile of the diode laser was controlled by a custom-written LabView template. The LED excitation intensity was kept constant for all experiments. Images were recorded with 2 fps using the imaging software AxioVision 4.8 (Zeiss).
Data evaluation. The recorded images were exported as TIFF files for data evaluation in ImageJ (US National Institutes of Health). The fluorescence intensity of donor (D) and acceptor (A) channel were readout for each pixel and averaged over the region of interest (ROI). Afterwards, the D/A ratio was calculated by dividing the donor by the acceptor intensity. The equilibrated D/A-value of each temperature jump (average of 2 frames at 18 s) is plotted against the temperature. We assume a two-state transition for RNA unfolding between folded, F, and unfolded, U, states. The fraction of the folded state is defined by: where K is the equilibrium constant for the unfolding reaction which is defined by: where [F] and [U] are the concentrations of the folded and unfolded states, respectively. To account for the intrinsic temperature dependence of both dyes Atto488 and Atto565 (Fig. S2) two linear baselines with the same slope representing the folded (a f +b(T-T min )) and unfolded (a u +b(T-T min ) state were used, respectively: The standard free energy of unfolding is defined by: Considering the temperature dependence of ΔG°u, the Gibbs-Helmholtz equation was used: where T m is the melting temperature, ΔH° the standard enthalpy change and ΔC°p the standard heat capacity change upon unfolding. The difference in heat capacity between the folded and unfolded states was neglected and Eq. (5) was simplified as: The standard free energy for unfolding, ∆°!, at T = 310 K (37 °C) was obtained by Fitting and student t-tests were carried out using Origin9 (OriginLab Corporation) and GraphPad Prism 6 (GraphPad Software), respectively.
If considering the equilibrium for RNA folding from a single strand accompanied by water uptake only, the number of water molecules released upon RNA unfolding Δn w was obtained using Eq. 8: 6 where R is the gas constant and T is the absolute temperature. Δn w and the error value were calculated from the slope of a linear fit weighted by the errors in ΔG°u. An analysis of covariance (ancova) reveals that the differences between the slopes obtained for sucrose and Ficoll 70 are not significant and thus an global slope was calculated with a correction coefficient r 2 >0.99. The dotted line for PEG in Figure 2A shows a correction coefficient r 2 >0.93. The numbers of water molecules released upon lm-4U RNA unfolding, Δn w , are 91±3 for sucrose/Ficoll and 33±5 for PEG < 6 kDa.
To verify the significant deviation for PEG 6 kDa and 20 kDa from the linear regression shown in Figure 2A, one sample t-tests with a 95% confidence interval against the interpolated free energy values at the experimentally determined water activities were performed. The p-values for PEG 6kDa and 20 kDa are p = 0.0063 and p = 0.0054, respectively. We further considered the error of a linear interpolation and tested the experimental values against the upper bound of the error. The difference was shown to be significant for PEG 6k (p=0.0113) and PEG 20k (p=0.0075) showing that PEG 6k and 20k differ significantly from the linear behavior of ln a w versus ΔG°u.
For single pixel evaluation, donor and acceptor images (TIFF) were background subtracted and aligned using ImageJ (NIH). For every time point, a D/A ratio map was generated by dividing donor images by the acceptor images. The TIFF files were then imported into Matlab and thermodynamic analysis was performed for every pixel. For every temperature step, 10 frames were averaged and the mean was used for thermodynamic fitting using Eq. (3). Only fits with errors of T m below 1 °C were used for the generation of the map. Typical fit errors in cells were in the range of 0.5±0.2 °C and therefore well below the observed variation. We further estimated the intrinsic error of the analysis by a comparative 30 x 30 px 2 region in vitro. From the single pixel analysis we obtained a melting temperature of 46.5 ± 0.1 °C and an unfolding free energy (at 37°C) of 5.3 ± 0.2 kJ/mol. We estimated the width of the variation by two standard deviations accounting for 95% of the variation. Thus, the intrinsic noise of the analysis falls within the range of ~ 0.2 k b T. The intracellular variations are therefore an order of magnitude above the experimental error. Histograms visualizing all single pixel measurements were extracted using ImageJ and then plotted using Graphpad Prism 6.
Gel electrophoresis and EtBr staining. The FReI temperature jump stepping was simulated in a PCR machine with a heat rate of 4 °C/s (T100 Thermal Cycler, Biorad). The RNA was purified by three consecutive extractions with phenol, phenol-chloroform, chloroform, respectively, and ethanol precipitation. 150 ng of the 4U RNA was separated on 10% polyacrylamide gels containing 7 M urea. RNAs were visualized by staining the gels with ethidium bromide.