Structural insights into nanoRNA degradation by human Rexo2

Abstract

Human RNA exoribonuclease 2 (Rexo2) is an evolutionarily conserved 3'-to-5' DEDDh-family exonuclease located primarily in mitochondria. Rexo2 degrades small RNA oligonucleotides of <5 nucleotides (nanoRNA) in a way similar to Escherichia coli Oligoribonuclease (ORN), suggesting that it plays a role in RNA turnover in mitochondria. However, how Rexo2 preferentially binds and degrades nanoRNA remains elusive. Here, we show that Rexo2 binds small RNA and DNA oligonucleotides with the highest affinity, and it is most robust in degrading small nanoRNA into mononucleotides in the presence of magnesium ions. We further determined three crystal structures of Rexo2 in complex with single-stranded RNA or DNA at resolutions of 1.8-2.2 Å. Rexo2 forms a homodimer and interacts mainly with the last two 3'-end nucleobases of substrates by hydrophobic and π-π stacking interactions via Leu53, Trp96, and Tyr164, signifying its preference in binding and degrading short oligonucleotides without sequence specificity. Crystal structure of Rexo2 is highly similar to that of the RNA-degrading enzyme ORN, revealing a two-magnesium-ion-dependent hydrolysis mechanism. This study thus provides the molecular basis for human Rexo2, showing how it binds and degrades nanoRNA into nucleoside monophosphates and plays a crucial role in RNA salvage pathways in mammalian mitochondria.

Keywords: RNA decay; crystal structure; exonuclease; protein–RNA interactions; ribonuclease.

FIGURE 1.

Rexo2 is a robust ribonuclease that degrades small RNA oligonucleotides into mononucleotides. (A) Recombinant Rexo2 purified to high homogeneity, as revealed by SDS-PAGE. (B) Rexo2 forms a homodimer with a MW of ∼44 kDa, as estimated by size exclusion chromatography-coupled multiangle light scattering (SEC-MALS). (C) The Rexo2–H194A mutant exhibits only residual RNase activity in degrading a 4-nt RNA relative to that of wild-type Rexo2. (D,E) Wild-type Rexo2 (1 µM) and E. coli ORN (50 nM) degrades 4-nt RNA (5′-³²P-A₄-3′) or 12-nt RNA (5′-³²P-A₁₂-3′) into mononucleotides in the presence of 5 mM MgCl₂, whereas the Rexo2–D199A mutant (1 µM) exhibits only residual RNase activity. (F–H) RNA binding affinities between Rexo2–H194A and 2-nt RNA (5′-Cy3-A₂-3′), 5-nt RNA (5′-Cy3-A₅-3′), and 11-nt RNA (5′-Cy3-AGCGCAGUACC-3′) substrates were measured by fluorescence polarization (in mFP units) and plotted against protein concentrations. The RNA-binding affinities of Rexo2 were calculated by fitting the binding curve to a one-site-binding Hill slope, giving estimated Hill coefficients of 2.3, 1.4, and 3.5 for 2-nt, 5-nt, and 11-nt RNA, respectively. See also Supplemental Figure S1.

FIGURE 2.

The crystal structures of the Rexo2–RNA and Rexo2–DNA complexes show how Rexo2 forms a homodimer and binds oligonucleotides. (A) The overall structure of the Rexo2–DNA2 complex. Rexo2 forms a homodimer, one protomer (chain B) displayed in green and the other (chain A) in blue, with one DNA strand and two Mg²⁺ ions bound in the active site of protomer B. (B) The electrostatic surface potential of the Rexo2–DNA2 complex reveals positive surfaces extending from the active site (red, −5.0 kBT/e; blue, +5.0 kBT/e; kB, Boltzmann constant; T, temperature in Kelvin; e, charge of an electron). (C–E) The omit electron density maps for the last two 3′-end nucleotides bound in the active site of protomer B in Rexo2–RNA complex (1.0 σ), Rexo2–DNA1 complex (0.8 σ), and Rexo2–DNA2 complex (1.0 σ). See also Supplemental Figures S2, S3.

FIGURE 3.

Rexo2 interacts with the last two 3′-end nucleobases of substrate by hydrophobic and π−π stacking interactions. (A–C) The last two 3′-end nucleobases are sandwiched between Tyr164, Leu53, and Trp96 in the Rexo2–RNA, Rexo2–DNA1, and Rexo2–DNA2 complexes via hydrophobic and π–π stacking interactions. (D) Schematic diagram for the interactions between Rexo2 and DNA in the Rexo2–DNA2 complex. The DNA-interacting residues in protomer B of Rexo2 are displayed in green, whereas the residue in protomer A (Arg165) is displayed in black. In the Rexo2–RNA complex, the hydrogen bond between 2′-OH (O2′) of the ribose group of the 3′-end nucleotide and the Met50 backbone (O atom) is displayed in red dashed line. See also Supplemental Figure S4.

FIGURE 4.

Rexo2 hydrolyzes RNA by a two-Mg²⁺-dependent mechanism. (A–C) Catalytic residues in the active site of Rexo2 in the crystal structures of Rexo2–RNA (A), Rexo2–DNA1 (B), and Rexo2–DNA2 (C). (D) Schematic diagram of the two metal-ion-dependent hydrolysis mechanism of Rexo2 responsible for degrading RNA from the 3′ end. The conserved DEDD residues, Asp47, Glu49, Asp147, and Asp199, coordinate two Mg²⁺ ions (Mg_A and Mg_B), whereas the general base His194 activates a water molecule (absent from the three structures) for nucleophilic attack of the scissile phosphate. See also Supplemental Movie S1.

FIGURE 5.

The crystal structure of Rexo2 strongly resembles that of ORN. (A) Crystal structure of Rexo2–RNA with the active-site residues and RNA shown in stick model. (B) Crystal structure of the apo form of ORN (PDBID :2IGI) from E. coli. (C) Crystal structure of RNase T bound with DNA (PDBID: 3NH1). (D) Superimposition of RNA-binding aromatic residues in the active site of Rexo2–RNA and ORN revealing that these residues are located at similar positions. (E) Stick representation of the two 3′-end DNA nucleobases (dGdG) that make π–π stacking interactions with the four aromatic Phe residues in the active site of RNase T.