Molecular mechanism of RNase R substrate sensitivity for RNA ribose methylation

Abstract

RNA 2'-O-methylation is widely distributed and plays important roles in various cellular processes. Mycoplasma genitalium RNase R (MgR), a prokaryotic member of the RNase II/RNB family, is a 3'-5' exoribonuclease and is particularly sensitive to RNA 2'-O-methylation. However, how RNase R interacts with various RNA species and exhibits remarkable sensitivity to substrate 2'-O-methyl modifications remains elusive. Here we report high-resolution crystal structures of MgR in apo form and in complex with various RNA substrates. The structural data together with extensive biochemical analysis quantitively illustrate MgR's ribonuclease activity and significant sensitivity to RNA 2'-O-methylation. Comparison to its related homologs reveals an exquisite mechanism for the recognition and degradation of RNA substrates. Through structural and mutagenesis studies, we identified proline 277 to be responsible for the significant sensitivity of MgR to RNA 2'-O-methylation within the RNase II/RNB family. We also generated several MgR variants with modulated activities. Our work provides a mechanistic understanding of MgR activity that can be harnessed as a powerful RNA analytical tool that will open up a new venue for RNA 2'-O-methylations research in biological and clinical samples.

Figure 1.

Enzymatic activity of MgR and its homologs. (A,B) RNA degradation assays of MgR and its homologs with substrates of 30-nt oligo rA without (A) or with (B) 2′-O-methylation at nucleotide rA15. The assays were performed at room temperature over time course of 0, 0.5, 1, 2, 4, 6, 8 or 16 min. Products were resolved by denaturing urea PAGE. The results at 4-min time point for each enzyme were collectively displayed. The uncropped gels with results of all the time points were shown in Supplementary Figures S2 and S3A. (C) Enzymatic activity (nucleotides (nt) min⁻¹ molecule⁻¹) of MgR compared with other homologs degrading the methylated substrate used in panel B and Supplementary Figure S3. Enzymatic activity was determined from the initial rate (pmol substrate degraded per minute) divided by the amount of enzyme (pmol) multiplied by (n-3 or n-15) (where n is the number of nucleotides of the substrate, 3 is the length of the end product, and 15 is the length of the intermediate product). (D) Intermediate productivity at 4 min time point of MgR compared with other homologs degrading methylated substrates used in panel B and Supplementary Figure S3. Intermediate productivity was determined using the initial intermediate product molar mass (pmol) divided by the total amount of RNA substrate molar mass (pmol) and multiplied by 100%. Mean ± S.D. are shown. (E) Mass spectrometry for the identification of MgR intermediate product. A 30-nt 5′-FAM labeled RNA substrate ((CAAAA)_n, n = 5) with a 2′-O-methylation at nucleotide rA15 was used. The schematic figures of the 15-nt and 16-nt intermediate products are illustrated and molecular weights (MW) marked. (F) TA clone sequencing for the identification of MgR intermediate product. A 40-nt ssRNA substrate with 2′-O-methylation at nucleotide rA28.

Figure 2.

Crystal structures of MgR and its RNA complex. (A) Overall structure of apo-MgR compared with MgR-ssRNA and the domain schematic. Apo-MgR (grey) is superimposed with MgR–ssRNA (domains colored). The 9-nt linear RNA substrate is colored in orange and each nucleotide from 5′ to 3′ is labeled. The Mg²⁺ ion is shown as green sphere. (B) Surface representation of MgR-ssRNA displaying the apical groove, lateral groove, and the active cavity in the RNB domain. The image has been clipped to facilitate observation of the cavity. The surface is colored according to domains as in panel A. The clipping plane is blanked in gray. (C) Overall structure of MgR–dsRNA. The ribbon is colored according to B-factors from blue with low values to red with high values. The missing part of the structure was drawn according to MgR–ssRNA and colored in grey.

Figure 3.

Critical RNA-interacting residues in the active cavity of MgR RNB domain. (A) A schematic view of the interactions between RNAs and the RNB domain of MgR. The dashed lines show the hydrogen bond interactions between rA5–rA9 (phosphate and ribose backbone in orange and 2′-hydroxyl groups in red) and side chains of residues (labeled ovals) in RNB domain. Mg²⁺ (colored in green) and structured water (colored in red) are labelled. (B–D) Structural conformational changes upon RNA binding by superimposing apo-MgR (in grey and residues marked with underlines) to MgR-ssRNA (in slate). Interactions between RNA ribose hydroxyls and surrounding residues are marked with dashed lines. RNA is shown as sticks in orange. Protein is shown as ribbon with side chains of related residues shown as sticks. Water molecules are shown as red spheres. Mg²⁺ is shown as green sphere. (E) Enzymatic activity (nt min⁻¹ molecule⁻¹) of various MgR mutants degrading a 30-nt rA with a 2′-O-methylation at nucleotide rA15. (F) Intermediate productivity (%) at 4-min time point of various MgR mutants degrading a 30-nt rA with a 2′-O-methylation at nucleotide rA15. The uncropped gels with results of all the time points were shown in Supplementary Figure S14.

Figure 4.

Molecular basis of MgR for recognition and significant sensitivity to RNA 2′-O-methylation. (A) The active cavity region of MgR is shown as grey surface and cartoon with side chains of residues P277 and H331 as sticks. The structure of MgR (slate) is superposed to those of EcR (cyan) and RNase II (yellow). The aligned EcR residue G273 and RNase II residue S202 are marked accordingly. A methyl group is modeled to the 2′-hydroxyl of rA8 to show the steric hindrance to MgR P277. (B) Mutagenesis studies on MgR P277 and EcR G273. Enzymatic activity assays were conducted on both the wild-types and mutants degrading RNA substrates with 2′-O-methylation. The intermediate productivity was determined as described in Figure 1C. (C) The structure-based sequence alignment including MgR, McR, MhR, EcR and E. coli RNaseII. The secondary structure elements (arrows for β strands and cylinders for α helices) and residue numbers of MgR are as shown. Those residues aligned to MgR P277 are highlighted in yellow. The conserved residues around the active site are highlighted in red. The sequences were aligned with Clustal Omega (64) and visualized using ESPript (65).