Structural basis of 3'-tRNA maturation by the human mitochondrial RNase Z complex

Abstract

Maturation of human mitochondrial tRNA is essential for cellular energy production, yet the underlying mechanisms remain only partially understood. Here, we present several cryo-EM structures of the mitochondrial RNase Z complex (ELAC2/SDR5C1/TRMT10C) bound to different maturation states of mitochondrial tRNA^His, showing the molecular basis for tRNA-substrate selection and catalysis. Our structural insights provide a molecular rationale for the 5'-to-3' tRNA processing order in mitochondria, the 3'-CCA antideterminant effect, and the basis for sequence-independent recognition of mitochondrial tRNA substrates. Furthermore, our study links mutations in ELAC2 to clinically relevant mitochondrial diseases, offering a deeper understanding of the molecular defects contributing to these conditions.

Keywords: Cryo-EM; ELAC2; Mitochondria; RNA Processing; RNase Z.

Figure 1. Structure of human mitochondrial RNase Z.

(A) Domain architecture of the human mitochondrial RNase Z components. The N-terminal (NTD) and C-terminal (CTD) domains of ELAC2 are shown in dark and light purple, respectively. MBL1 and MBL2, which are the metallo-β-lactamase folds, and the exosite insertion are indicated with dotted lines. TRMT10C is shown in blue, with the NTD highlighted in cyan. MTS, mitochondrial targeting sequence. Unmodelled regions are in white. (B) The mt-tRNA precursor consists of tRNA^His (red) and tRNA^Ser(AGY) (gray line), of which the latter has been cleaved off in the ternary product structure. Base-paired nucleotides are in red circles, and non-base-paired ones are in white circles. The anticodon triplet is in gray circles. Canonical tRNA numbering is indicated (Giegé et al, 2012) and is used throughout the text (the correspondence with nucleotide numbers in mitochondrial tRNA^His, which was used in the PDB deposited structures, is shown in Appendix Table S1). (C, D) Composite cryo-EM density (C) and cartoon representation (D) of the RNase Z complex. Colored as in A. The four SDR5C1 subunits are shown in different shades of gray. The ELAC2 active site is marked with a yellow star.

Figure 2. Stabilization of the tRNA fold by TRMT10C.

(A) TRMT10C (white) wraps around the mt-tRNA^His anticodon stem and D arm to stabilize the cloverleaf fold. The nucleotide numbers forming the T loop are indicated for comparison with those in (B, C). (B, C) Molecular interactions of TRMT10C with the T loop (B) and the D arm (C). The views have been rotated for easier visualization.

Figure 3. Interaction of ELAC2 with the precursor mt-tRNA^His.

(A) Overview of the human mitochondrial RNase Z complex. The boxes represent the insets for (B–E). (B) Contacts between the ELAC2 exosite, the T loop, and the TRMT10C N-terminal domain. (C) Contacts between the ELAC2 C-terminal helix, the TRMT10C, and the acceptor stem. (D) Recognition of the 5′-end of the mt-tRNA. Only the α-phosphate of G1 is shown since natural ELAC2 substrates carry a 5′-monophosphate. (E) Interaction of ELAC2 with the acceptor stem/T arm groove.

Figure 4. Interactions around the tRNA scissile bond and the ELAC2 HFSQRY motif.

(A) In the RNAse Z-HS structure, the 3′-oxygen interacts with a Zn²⁺ after the 3′-trailer is removed (B) Composite cryo-EM density of the human mitochondrial RNase Z bound to the mt-tRNA^His-Ser(AGY) precursor, carrying the ELAC2 mutation H548A. The part of the map corresponding to mt-tRNA^Ser(AGY) moiety was gaussian-filtered with a B-factor of 600 in UCSF ChimeraX. Colored as in Fig. 1. (C) The RNase Z^H548A-HS structure shows how the scissile bond is well-oriented for catalysis assisted by K700. The missing Zn²⁺ due to the H548A mutation is shown by a transparent circle based on the RNase Z-HS structure. In the RNase Z-HS structure, the 3′-oxygen interacts with a Zn²⁺ after the 3′-trailer is removed.

Figure 5. Structural basis for 3′-CCA discrimination.

(A) ELAC2 dilution series on 200 nM mt-tRNA^His-GAG or mt-tRNA^His-CCA and 800 nM TRMT10C/SDR5C1 was analyzed using a TBE-UREA PAGE. Representative gel image of technical triplicates and biological duplicates for WT ELAC2. (B) Composite cryo-EM density of human mitochondrial RNase Z bound to the mt-tRNA^His-CCA. Colored as in Fig. 1. The density for the 3′-CCA trailer is indicated with a yellow circle. (C) The 3′-CCA occupies the active site channel of ELAC2. The mt-tRNA^His 3′-end is 13.4 Å away from the catalytic Zn²⁺ (gray spheres). (D) Interactions of C1^CCA with ELAC2 S490 and S726. (E) Interactions of C2^CCA with Q92 and T127. The hydrogen atoms were added in ChimeraX to visualize the H-bonding interactions. (F) Comparison with the tRNA^Thr precursor bound to the B. subtilis RNase Z (Pellegrini et al, 2012), which shows the interactions required for catalysis. The phosphate oxygens coordinate one Zn²⁺ ion each. The hydrolyzing water molecule (red sphere) is positioned by D550. In RNase Z-HCCA, the backbone adopts a different conformation, which is unsuitable for catalysis. Source data are available online for this figure.

Figure EV1. The TRMT10C structure (related to Fig. 1).

(A) ELAC2 activity in the presence and absence of the TRMT10C/SDR5C1 platform. (B) Cryo-EM density of particles containing two TRMT10C subunits. The 4xSDR5C1 platform offers two mt-tRNA/TRMT10C binding sites. The platform has a vertical and a horizontal twofold symmetry axis. Consequently, it supports two mt-tRNA/TRMT10C subcomplexes, which can be in two different but equivalent orientations (left and right panels). The 4xSDR5C1 are colored in different shades of gray; TRMT10C is colored in dark blue with the NTD highlighted in light blue; the mt-tRNA is in red. ELAC2 density is not visible due to the low occupancy. Source data are available online for this figure.

Figure EV2. TRMT10C/SDR5C1 interactions with ELAC2 and PRORP (related to Fig. 2).

(A) ELAC2 cleavage of the mt-tRNA^His-GAG precursor in the absence of TRMT10C/SDR5C1, or with the mutations V256E_L257E that affect residues interacting with TRMT10C. (B) TRMT10C NTD interacts with ELAC2 (RNase Z, left panel) and PRORP (RNase P, right panel) in the T loop region. (C) The ELAC2 C-terminal helix would collide with a 5′ extension on the mt-tRNA precursor (yellow circle). To make this figure, the mt-tRNA precursor in RNase Z was replaced with the RNase P one (mt-tRNA^Tyr), which has a 2-nucleotide 5′-extension. For this, The RNase Z structure was superimposed on the RNase P structure (Bhatta et al, ; PDB 7ONU), using TRMT10C as a reference. Source data are available online for this figure.

Figure EV3. Pivoting of ELAC2 on the mt-tRNA (related to Fig. 3).

(A) ELAC2 pivots on the mt-tRNA to reach the catalytic position. The RNase Z-HS structure is at the forefront. The outline of the RNase Z^H548A-HS and the RNase Z-HCCA structures are shown to highlight the movement of ELAC2. ELAC2 is colored in light and dark purple for the CTD and NTD, respectively, while TRMT10C is in blue, and the four SDR5C1 subunits are in shades of gray. (B) Cryo-EM composite maps of RNase Z bound to the three different tRNA precursors, aligned on TRMT10C. The cryo-EM density of the mt-tRNA^Ser(AGY) moiety has been Gaussian-filtered in UCSF ChimeraX with a B-factor of 600 for better visualization. To visualize the ELAC2 movement, notice the changes in the gap with TRTM10C. (C) Cryo-EM density for the mt-tRNA^Ser(AGY) in RNase Z^H548A-HS structure. The density, for which it was not possible to create an atomic model, displaces the G157-P157 loop compared with the RNase Z-HS structure (superposed in white). (D) Comparison with the B. subtilis RNase Z bound to precursor tRNA. In B. Subtilis (white), U1 inserts into a pocket and helps position the scissile bond. In RNase Z^H548A-HS, G1 does not bind in the pocket as it is partially occluded by L126. The B. Subtilis Zn²⁺ are shown as white spheres. The RNase Z^H548A-HS missing Zn²⁺ is drawn based on the RNase Z-HS structure.

Figure EV4. Selected ELAC2 mutations involved in hypertrophic cardiomyopathy do not impair the 3’-CCA antidetermination (related to Fig. 5).

(A) Interactions around the tRNA 3′-end or the 3′-CCA tail involve the same ELAC2 residues. Two parallel black lines indicate π-stacking. Selected electrostatic interactions are indicated with gray dotted lines. (B) Selected clinical mutations near the ELAC2 active site that only mildly impact the catalytic activity, namely T520I (Haack et al, 2013), F154L (Haack et al, 2013), and G132R (Paucar et al, 2018). Mutations are shown in dark and light purple for the NTD and the CTD of ELAC2, respectively. The rest of ELAC2 is in gray, and the tRNA is in red. (C) Cleavage activity of the ELAC2 mutants on tRNA^His-CCA assessed by TBE-UREA PAGE. None of the mutations significantly affects the 3′-CCA antideterminant effect. The WT panel is the same as in Fig. 5A, shown here for an easier side-to-side comparison. Representative gel images of technical triplicates and biological duplicates for WT. Source data are available online for this figure.