Figure 1.
Schematic representation and structure of Dicer-dsRBD.
(A) Schematic representation of the primary sequence of Dicer with approximate location of domains above the cartoon. The primary sequence of human Dicer-dsRBD (residues 1850 to 1922) utilized in this study is shown in the box below the schematic, with the approximate location of secondary elements shown above the sequence(H represents an α-helix, B a β-sheet, and L a loop/turn). (B) Cryo-EM reconstruction is shown with the surface colored gray and the individual domains colored the same as in the above schematic. The tertiary structure of the mouse Dicer-dsRBD (pdb 3C4B, residues 1833 to 1900), which is 100% identical to the human sequence (residues 1849 to 1916), is depicted in the box to the right of the cryo-EM reconstruction.
Figure 2.
EMSA of pre-mir-16-1 and ds44 binding by Dicer-dsRBD.
EMSA of Dicer-dsRBD binding (A) pre-mir-16-1 with a Kd = 2.2±0.1 µM and (B) ds44 with a Kd = 2.4±0.1 µM. The predicted secondary structures of the RNAs are shown above the representative gels, which were run with varying Dicer-dsRBD concentration (0.25–50.12 µM) binding to 0.125 nM RNA. Fraction bound, from the EMSA data, versus Dicer-dsRBD concentration was fitted using a generalized Hill model (gray line). Error bars in the plots of fraction bound as a function of total Dicer-dsRBD concentration represent the standard deviation from duplicate measurements.
Table 1.
Binding Affinity of Dicer-dsRBD in Isolation for Various Length RNA Constructs by EMSA.
Figure 3.
Sedimentation velocity analysis of Dicer-dsRBD binding to ds16.
Plots of normalized g(s*) distributions for 2.0 µM ds16 alone (black) and 2.0 µM ds16 plus 4.3 µM (red), 8.5 µM (blue), 21.4 µM (green), and 42.7 µM (lavender) Dicer-dsRBD. The distributions are normalized by area. The shift in the peak position from 2.3 S for the RNA alone to 3.3 S for the complex corresponds to a one-to-one binding stoichiometry. The data were globally analyzed using a one-to-one binding model to yield a best fit Kd = 5.4±0.7 µM, which agrees well with the EMSA data.
Figure 4.
EMSA of ds16-tetra-stable and ds16-octa-U binding by Dicer-dsRBD.
EMSA of Dicer-dsRBD binding (A) ds16-tetra-stable with a Kd = 9.1±0.1 µM and (B) ds16-octa-U with a Kd = 4.7±0.1 µM. The predicted secondary structures of the RNAs are shown above the representative gels, which were run with various Dicer-dsRBD concentration (0.25–50.12 µM) binding to 0.125 nM RNA. Fraction bound, from the EMSA data, versus Dicer-dsRBD concentration was fitted using a generalized Hill cooperative model (gray line). Error bars in the plots of fraction bound as a function of total Dicer-dsRBD concentration represent the standard deviation from duplicate measurements.
Table 2.
Effect of Hairpin Structure on the Binding Affinity of Dicer-dsRBD for dsRNA.
Figure 5.
NMR titration of Dicer-dsRBD with ds33.
Representative 15N-HSQC spectra of Dicer-dsRBD collected in the unbound state and in the presence of ds33. (A) Reference spectrum of apo-Dicer-dsRBD. (B) Ratio of individual peak intensities in the presence of 0.02∶1 mole ratio ds33:Dicer-dsRBD to those recorded under identical conditions in the apo-state spectrum displayed in (A). (C) Representative spectra from the ds33 titration showing the data points with mole ratios of 0.02∶1, 0.20∶1, and 2.0∶1 ds33:Dicer-dsRBD as labeled. All spectra were collected at 25°C in the presence of 100 mM KCl on a spectrometer operating at 600 MHz field strength.
Figure 6.
15N spin relaxation experiments of Dicer-dsRBD.
15N spin relaxation data for Dicer-dsRBD collected at 500 MHz (purple) and 600 MHz (gray) shows that the most dynamic regions of Dicer-dsRBD on the picosecond to nanosecond timescale are the loops, most notably loop 4. The secondary elements are represented as purple bars above the plot.
Figure 7.
Order parameters and RMSD of Dicer-dsRBD along with PDB bundle.
(A) Order parameters (S2) plotted for Dicer-dsRBD show that the most flexible regions in the protein are loops 3 and 4. Experimental data (purple) is plotted with MD predicted order parameters (gray). The secondary elements are represented as purple bars above the plot. (B) The overall stability of Dicer-dsRBD during the 250 ns MD simulation is demonstrated by the low average RMSD (<1.0 Å). (C) MD-derived ribbon bundle for Dicer-dsRBD also shows the stability of the construct. Increased flexibility, derived from the experimental order parameters, is depicted colorimetrically on the ribbon bundle as passage from purple (high order parameter) to yellow (low order parameter). The bundle was created by taking structures from the simulation every 50 ns and superimposing them to remove translational and rotational motion.
Figure 8.
Cα correlations of Dicer-dsRBD.
The Cα correlation matrix of Dicer-dsRBD reveals backbone motions. The color bar on the right shows the scale indicating strong positive correlation (red), strong negative correlation (blue), and non-correlated (green) motions. Labels above the panel indicate the location of secondary structural elements within the sequence.