Novel Insights into Guide RNA 5'-Nucleoside/Tide Binding by Human Argonaute 2

Abstract

The human Argonaute 2 (hAgo2) protein is a key player of RNA interference (RNAi). Upon complex formation with small non-coding RNAs, the protein initially interacts with the 5'-end of a given guide RNA through multiple interactions within the MID domain. This interaction has been reported to show a strong bias for U and A over C and G at the 5'-position. Performing molecular dynamics simulations of binary hAgo2/OH-guide-RNA complexes, we show that hAgo2 is a highly flexible protein capable of binding to guide strands with all four possible 5'-bases. Especially, in the case of C and G this is associated with rather large individual conformational rearrangements affecting the MID, PAZ and even the N-terminal domains to different degrees. Moreover, a 5'-G induces domain motions in the protein, which trigger a previously unreported interaction between the 5'-base and the L2 linker domain. Combining our in silico analyses with biochemical studies of recombinant hAgo2, we find that, contrary to previous observations, hAgo2 is capable of functionally accommodating guide strands regardless of the 5'-base.

Keywords: MD; RNAi; enzyme kinetics; fluorescence spectroscopy; pre-steady-state kinetics.

Figure 1

Analysis of backbone RMSD as a function of the simulation time for various hAgo2/guide RNA complexes. RMSD of the individual domains are calculated after superimposition on backbone atoms of the PIWI domain. (a) X-ray structure of full-length hAgo2 in complex with a truncated 10-mer guide miRNA (PDB 4F3T). The individual domains are labeled and color-coded: N (blue), PAZ (magenta), MID (gold) and PIWI (green) joined by two linker regions, L1 (silver) and L2 (pale cyan). The protein is represented in cartoon and the guide RNA in licorice; (b) Free hAgo2 in the absence of bound guide RNA; and hAgo2–guide RNA complexes with U (c); A (d); C (e); or G (f) at the 5′-end of the RNA.

Figure 2

PCA analysis on backbone atoms of hAgo2/guide RNA complexes with different 5′-bases. For clarity, the nucleic acid is not shown. (a) U; (b) A; (c) C; and (d) G at the 5′-position of the guide RNA. Color-coding is given in Figure 1.

Figure 3

Cross-RMSD plots of hAgo2 MID (a); and N-terminal (b) domain. Cross-RMSD were calculated after superimposition on backbone atoms of the MID and N domains. The different 5′-bases are indicated on the left. While the RMSDs (color-coded with a gradient from 0 to 5 Å) reveal very little changes in the MID domain between the various 5′-end guides, a clear difference can be observed in the N-terminal domain with gradually increasing RMSDs when switching form 5′-U to 5′-G end guides.

Figure 4

Fluorescence equilibrium titrations of hAgo2 with guide RNA. 20 nM of FAM-labeled guide RNA carrying a 5′-G (s2b-FAM) without (a) or with (b) a phosphate group were titrated with increasing amounts of hAgo2. Data were fitted to a quadratic equation and a representative fit is shown. K_ds of 7.5 ± 1.8 nM without a phosphate group and 34.7 ± 2.8 nM with a phosphate group at the guide 5′-end were obtained.

Figure 5

hAgo2-mediated cleavage of target RNA with guide strands carrying either a 5’-uridine or a 5’-guanosine with or without a 5′-phosphate. Cleavage assays were conducted using 2.5 µM hAgo2, 100 nM guide RNA (as2b or G-as2b) and 2.5 nM target RNA (ICAM-1-IVT). Samples were taken at time points 0ʹ, 5ʹ, 25ʹ, 55ʹ, and 120ʹ and analyzed using 8% denaturing PAGE. Detection was carried out via autoradiography.

Figure 6

Close-up of the guide RNA 5'-end and relevant protein residues of the MID domain and L2 linker region. The MID domain and the L2 helix are represented in cartoon (grey); important protein residues (green); and 5′-U (yellow); or 5′-G (blue) are shown in sticks. For clarity, merely the terminal base is shown. Hydrogen bonds are represented by black dotted lines. Transparent spheres highlight stacking interactions between protein residues and the corresponding bases. (a,d) show the initial situation; and (b,e) after 20 ns of simulation of 5′-U and 5′-G terminated hAgo2 bound guide strands, respectively; (c,f) superposition of (a,b); and (d,e) with neighboring protein residues at t = 0 ns are shown in green and at t = 20 ns in magenta.