Arginine Methylation Regulates Ribosome CAR Function

Abstract

The ribosome CAR interaction surface is hypothesized to provide a layer of translation regulation through hydrogen-bonding to the +1 mRNA codon that is next to enter the ribosome A site during translocation. The CAR surface consists of three residues, 16S/18S rRNA C1054, A1196 (E. coli 16S numbering), and R146 of yeast ribosomal protein Rps3. R146 can be methylated by the Sfm1 methyltransferase which is downregulated in stressed cells. Through molecular dynamics analysis, we show here that methylation of R146 compromises the integrity of CAR by reducing the cation-pi stacking of the R146 guanidinium group with A1196, leading to reduced CAR hydrogen-bonding with the +1 codon. We propose that ribosomes assembled under stressed conditions have unmethylated R146, resulting in elevated CAR/+1 codon interactions, which tunes translation levels in response to the altered cellular context.

Keywords: A-site decoding center; arginine methylation; codon adjacency; mRNA GCN periodicity; molecular dynamics; ribosome translocation; stress regulation.

Figure 1

The CAR interaction surface. (A) Cryo-EM structure (PDB ID 5JUP; [17]) of translocation stage II yeast ribosome showing large (yellow) and small (grey) subunits. Additionally, highlighted in color are the A site codon/anticodon, CAR interface, and +1 codon next to enter the A site. (B) Cartoon showing interaction between the CAR interface and first (G1) and second (C2) nucleotides of the +1 codon. H-bond donors are illustrated in red and H-bond acceptors in blue. (C) Rps3 R146 is hypothesized to be ω-N^G-monomethylated (arrow) in unstressed cells, and unmethylated in stressed cells. (D) van der Waal sphere representation of CAR interface showing stacking between the R146 guanidinium group and the A1196 base of the CAR interaction surface. Atoms are colored: N (blue), C (cyan), O (red), H (white), P (olive).

Figure 2

Methylated R146 shows elevated fluctuation. RMSF (Å) was calculated for residues comparing 30 trajectories with methylated (mR146) and unmethylated (R146) arginine. RMSF is shown for residues: CAR (C1054, A1196, (m)R146), +1 codon (+1 G1, +1 C2, +1 U3), A-site codon (A-C1, A-C2, A-U3) and tRNA anticodon (nt36, nt35, nt34). (A) shows RMSF calculated with “core” heavy atoms of bases (C2, C4, C5, C6, N1, N3 of C and U; C2, C4, C5, C6, C8, N1, N3, N7, N9 of A and G) and the guanidinium group (CZ, NE, NH1, NH2). mR146 has significantly greater fluctuations than R146 (t-test p < 0.001 ***). (B) shows RMSF for backbone heavy atoms. No significant differences between the mR146 and R146 trajectories were observed in measurements of backbone RMSF. (C) Illustration of core and backbone atoms used for RMSF.

Figure 3

R146 methylation disrupts cation-pi stacking between R146 and A1196 in the CAR interface. (A) Stacking was assessed by measuring the distances between the centers of mass of the heavy atoms of bases or the guanidinium group of R146. Center of mass (COM) distances were measured for anticodon nt35 to nt34, anticodon nt34 to C1054, C1054 to A1196, and A1196 to R146, comparing the trajectories for methylated (mR146) and unmethylated (R146) guanidinium group. 30 trajectories were compared for each. The center of mass distance is significantly higher for mR146-A1196 compared to R146-A1196 (t-test p < 0.01 **). (B) A stripped version of the solvent accessible surface area (SASA) protocol (see Materials and Methods) was used to assess solvent accessibility between (m)R146 and A1196 (in Ų). The methylated guanidinium group had significantly higher solvent accessibility than unmethylated (t-test p < 0.01 **), suggesting that the methylation disrupts cation-pi stacking with A1196.

Figure 4

R146 methylation reduces CAR/+1 codon H-bonding. H-bond interactions were quantitated for the A-site nucleotides 2 and 3 and the three +1 codon nucleotides with unmethylated (1.6 µs) and methylated (1.6 µs) R146. All hydrogen bonds were counted between these residues and the anticodon and CAR residues (e.g., interactions of C2 with C1054, A1196 and R146). (A) Overall H-bond counts show significantly depressed H-bonds for the second nucleotide of the +1 codon (C2) comparing methylated and unmethylated R146 trajectories (t-test p < 0.001 ***). (B) Illustrated are all H-bond interactions with frequencies f: 0.025 < f < 0.095 (grey dashes) or f > 0.095 (red dashes). This reveals depressed H-bond interactions for methylated R146 compared to unmethylated.

Figure 5

Comparisons across trajectory experiments. (A) 20 independently-initiated trajectories were subjected to pairwise RMSD comparisons (between all pairs of frames) using backbone heavy atoms (bottom right-hand triangles below the diagonals) or “core” base and guanidinium group heavy atoms (top left-hand triangles) for 12 residues: CAR (C1054, A1196, (m)R146), +1 codon (+1 G1, +1 C2, +1 U3), A site (A-C1, A-C2, A-U3) and tRNA anticodon (nt36, nt35, nt34). The methylated R146 subsystem had a greater difference between RMSD of backbone and core atoms (right) compared to unmethylated R146 (left). Each trajectory was sampled with 41 frames (1 frame per ns). (B) For each of the 20 trajectories, the average RMSD difference with the other 19 trajectories was calculated. The differences (in Å) between the core and backbone atom RMSD means (averaged by experiment) were graphed. Bootstrap analysis indicated that mR146 trajectories had a significantly greater difference than for R146 (12 residue; p < 0.001 ***; 10,000 bootstrap samples). This was also true when the R146 residue was excluded from the RMS2D calculations (11 residue; p < 0.01 **).

Figure 6

Arginine methylation regulates ribosome CAR function. Methylation of Rps3 R146 is hypothesized to downregulate CAR function by reducing cation-pi stacking of the R146 guanidinium group with A1196 which in turn reduces H-bonding of CAR with the +1 codon next to enter the ribosome A site. Expression of Sfm1, the methyltransferase responsible for R146 methylation, is down regulated in response to stress, promoting CAR integrity and CAR/+1 codon H-bonding.