MD Simulations of the dsRBP DGCR8 Reveal Correlated Motions that May Aid pri-miRNA Binding

Abstract

Over the past decade, microRNAs (miRNAs) have been shown to affect gene regulation by basepairing with messenger RNA, and their misregulation has been directly linked with cancer. DGCR8, a protein that contains two dsRNA-binding domains (dsRBDs) in tandem, is vital for nuclear maturation of primary miRNAs (pri-miRNAs) in connection with the RNase III enzyme Drosha. The crystal structure of the DGCR8 Core (493-720) shows a unique, well-ordered structure of the linker region between the two dsRBDs that differs from the flexible linker connecting the two dsRBDs in the antiviral response protein, PKR. To better understand the interfacial interactions between the two dsRBDs, we ran extensive MD simulations of isolated dsRBDs (505-583 and 614-691) and the Core. The simulations reveal correlated reorientations of the two domains relative to one another, with the well-ordered linker and C-terminus serving as a pivot. The results demonstrate that motions at the domain interface dynamically impact the conformation of the RNA-binding surface and may provide an adaptive separation distance that is necessary to allow interactions with a variety of different pri-miRNAs with heterogeneous structures. These results thus provide an entry point for further in vitro studies of the potentially unique RNA-binding mode of DGCR8.

The Core Microprocessor Component DiGeorge Syndrome Critical Region 8 (DGCR8) Is a Nonspecific RNA-binding Protein

“…6). This demonstrates that the two tandem RBD domains interact with each other in solution confirming earlier reports based on the crystal structure (49) and MD simulations (54).…”

“…Instead, noticeable broadening occurs within the ␤4, ␤7, and ␣7 regions. For its part, ␣7 has been shown to stabilize the DGCR8 core through extensive intramolecular contacts with both dsRBDs (49,54). Therefore, line broadening in this region is not likely attributed to direct association with pri-mir-16 lower but is probably reporting on conformational exchange on an intermediate time scale between bound and free DGCR8 core forms.…”

The Role of Human Dicer-dsRBD in Processing Small Regulatory RNAs

“…Root-mean-square deviation (RMSD) from the starting structure over the course of the trajectories verified that the Dicer-dsRBD was stable over the 250 ns simulation (Figure 7B ). The very low 1.0 Å RMSD seen for a large majority of the simulation is the lowest RMSD we have reported for any dsRBD in isolation, [23], [42] highlighting the high stability of the backbone of Dicer-dsRBD as compared with other dsRBDs. Further evidence of the stability of the Dicer-dsRBD comes from ribbon bundles (Figure 7C ) of the simulations overlapping well and showing no loss in secondary structure elements.…”

“…Simulations were carried out in explicit solvent represented by the SPC water model [40] under particle mesh Ewald periodic boundary conditions. [41] Dicer-dsRBD MD simulations were run as previously reported [42] using the crystal structure (3C4B, residues 1833–1900 of the mouse sequence corresponding with 1849–1916 of the human sequence). Nine chloride counterions were added to neutralize the net positive charge on the protein, and then the resulting system was solvated such that no solute atom was within 10 Å of a box edge, requiring 7031 water molecules.…”

Binding by TRBP-dsRBD2 Does Not Induce Bending of Double-Stranded RNA

“…Although this precludes a quantitative comparison of our simulation results with the original crystal structures, the qualitative trends revealed by the simulations are expected to have merit. Before the production runs, both systems were energy minimized and equilibrated as previously described (27,28). After equilibration, production simulations were performed in the NPT ensemble for 250 ns per system, with a 2 fs time step and a frame save-down rate of 1/ps.…”