Cleavage kinetics of human mitochondrial RNase P and contribution of its non-nuclease subunits

Abstract

RNase P is the endonuclease responsible for the 5' processing of precursor tRNAs (pre-tRNAs). Unlike the single-subunit protein-only RNase P (PRORP) found in plants or protists, human mitochondrial RNase P is a multi-enzyme assembly that in addition to the homologous PRORP subunit comprises a methyltransferase (TRMT10C) and a dehydrogenase (SDR5C1) subunit; these proteins, but not their enzymatic activities, are required for efficient pre-tRNA cleavage. Here we report a kinetic analysis of the cleavage reaction by human PRORP and its interplay with TRMT10C-SDR5C1 including 12 different mitochondrial pre-tRNAs. Surprisingly, we found that PRORP alone binds pre-tRNAs with nanomolar affinity and can even cleave some of them at reduced efficiency without the other subunits. Thus, the ancient binding mode, involving the tRNA elbow and PRORP's PPR domain, appears basically retained by human PRORP, and its metallonuclease domain is in principle correctly folded and functional. Our findings support a model according to which the main function of TRMT10C-SDR5C1 is to direct PRORP's nuclease domain to the cleavage site, thereby increasing the rate and accuracy of cleavage. This functional dependence of human PRORP on an extra tRNA-binding protein complex likely reflects an evolutionary adaptation to the erosion of canonical structural features in mitochondrial tRNAs.

Graphical Abstract

Figure 1.

Human PRORP has RNase P activity without TRMT10C-SDR5C1. The RNase P activity of PRORP was tested on model substrates of the six indicated human mitochondrial pre-tRNAs. Aliquots were withdrawn from the reactions after 3, 30 and 60 min for pre-tRNA^Ala and pre-tRNA^Val, and 10, 30 and 60 min for pre-tRNA^His, pre-tRNA^Ile, pre-tRNA^Lys and pre-tRNA^Met; cleavage products were separated by gel electrophoresis and visualized by phosphorimaging. The final concentration of the TRMT10C–SDR5C1 complex was 250 nM. No enzyme was added to the ‘mock’ reaction, which was incubated for 60 min. Due to 5′-end labeling, only the full-length pre-tRNA and the released 5′ leader are visible. (A) Examples of mitochondrial pre-tRNAs whose 5′-end processing requires both, PRORP and the TRMT10C–SDR5C1 complex, or (B) on which PRORP alone showed RNase P activity in vitro.

Figure 2.

(Mis-)cleavage of an RNase P reference substrate by human PRORP. The RNase P activity of human PRORP was tested on bacterial T. thermophilus (Tt) pre-tRNA^Gly. Aliquots were withdrawn from the reactions after 3, 30 and 60 min, and analyzed as described for Figure 1. A processing reaction with A. thaliana PRORP3 (At PRORP3) was included to identify the position of the canonical cleavage site between +1 and −1. In the first and last lanes, a nucleotide-resolution ladder, generated from pre-tRNA^Gly by partial alkaline hydrolysis (AH), was loaded to determine whether the observed extra cleavage occurred one or two nucleotides upstream of the canonical cleavage site. (Mis-)cleavage between pre-tRNA nucleotides −1 and −2 resulted in a one-nucleotide shorter 5′-leader product.

Figure 3.

TRMT10C-SDR5C1 inhibits the activity of ELAC2 and A. thaliana PRORP3, and cannot be replaced by other TRM10 methyltransferases in mtRNase P reactions. (A) The effect of the TRMT10C–SDR5C1 complex on the RNase Z activity of ELAC2 was tested. Mitochondrial pre-tRNA^Ala with mature 5′ end was supplemented with the indicated concentrations of TRMT10C–SDR5C1 and tested for 3′ cleavage by His-ELAC2 (25 nM); aliquots were withdrawn at 1, 5 and 60 min, and analyzed as described for Figure 1. (B) The ability of the TRMT10C-SDR5C1 complex to stimulate RNase P cleavage was tested with a plant PRORP homolog. Human mitochondrial pre-tRNA^His was supplemented with the indicated concentrations of human TRMT10C–SDR5C1, and tested for cleavage by A. thaliana PRORP3 (At PRORP3; 100 nM); aliquots were withdrawn at 3, 30, and 60 min, and analyzed as described for Figure 1. (C) The RNase P activity of human PRORP (25 nM) was tested on human mitochondrial pre-tRNA^Ile in the presence of different methyltransferases of the TRM10 family: the human mitochondrial TRMT10C-SDR5C1 complex, S. cerevisiae Trm10p (Sc Trm10p), and human nuclear TRMT10A, each at a final concentration of 250 nM. Aliquots were withdrawn after 0.25, 15 and 60 min, and analyzed as described for Figure 1.

Figure 4.

Single-turnover kinetics of pre-tRNA cleavage by the mtRNase P holoenzyme and human PRORP alone. Single-turnover kinetic analyses of pre-tRNA cleavage were performed with varying concentrations of PRORP in the presence of an excess of TRMT10C–SDR5C1 complex (mtRNase P holoenzyme; panels A, B, E and F) or PRORP alone (panels C and D). Pseudo first-order rate constants of cleavage (k_obs or k_obs*; see Materials and Methods, and Supplementary Figure S3 and S4) were plotted against the concentration of PRORP. Data points are the mean ± SEM of at least five replicates. Derived kinetic constants k_react and K_M(sto) (best-fit values ± curve-fit standard error) are inserted into each graph. (A, B) Kinetic analysis of the cleavage of (A) pre-tRNA^Ala (at 3 mM Mg²⁺) and of (B) pre-tRNA^Met (at 4.5 mM Mg²⁺) by PRORP in the presence of TRMT10C–SDR5C1. (C, D) Kinetic analysis of the cleavage of (C) pre-tRNA^Ala (at 3 mM Mg²⁺) and of (D) pre-tRNA^Met (at 4.5 mM Mg²⁺) by PRORP alone. (E, F) Kinetic analysis of the cleavage of (E) pre-tRNA^Ile (at 3 mM Mg²⁺) and of (F) pre-tRNA^Lys (at 4.5 mM Mg²⁺) by PRORP in the presence of TRMT10C–SDR5C1.

Figure 5.

Metal-ion coordination in the active site of human PRORP. (A) PRORP was subjected to iron-mediated hydroxyl radical cleavage. The wild-type protein, or substitution variants D479N and D499N, were incubated with Fe(II), DTT and/or ascorbic acid, and cleavage products were resolved by SDS-PAGE; the gel was stained with Coomassie blue. Specific cleavage products in lanes 4–6 are indicated by asterisks (typically, only a small fraction of the protein is cleaved by this procedure, as previously demonstrated for other, well-established metalloenzymes; refs. 43,63); the barely visible ∼20 kDa fragment and the second, smaller complementary C-terminal fragment (not detectable by direct staining) were confirmed by western blotting (Supplementary Figure S10). The molecular weight of selected size markers (lane 1) is indicated on the left. The position of full-length PRORP is indicated on the right. (B) PRORP was subjected to iron-mediated hydroxyl radical cleavage in the presence of pre-tRNA^Ile (lane 4) and/or the TRMT10C-SDR5C1 complex (lanes 7 and 6). Specific cleavage products in lanes 3, 4 and 6, 7, are indicated by asterisks (note that the ∼40-kDa PRORP fragment migrates very close to TRMT10C in lanes 6 and 7, and can only be seen when zooming into the relevant area). The positions of full-length PRORP, TRMT10C and SDR5C1 are indicated on the right.

Figure 6.

Mg²⁺ optimum of human PRORP compared to that of the mtRNase P holoenzyme. Cleavage kinetics were performed with PRORP alone and in the presence of TRMT10C-SDR5C1 (mtRNase P holoenzyme), at different Mg²⁺ concentrations under single-turnover conditions. Pseudo first-order cleavage rates (k_obs or k_obs*) were derived and normalized to the highest rate measured. Relative rates represent the mean ± SEM of at least three replicates. Cleavage by PRORP alone at the lowest and/or highest Mg²⁺concentration shown for the mtRNase P holoenzyme was in several cases too weak to derive a corresponding cleavage rate, explaining the missing data points. (A–C) Mg²⁺ optima for the cleavage of (A) pre-tRNA^Ala, (B) pre-tRNA^Glu and (C) pre-tRNA^Met.

Figure 7.

Multiple-turnover kinetics of pre-tRNA cleavage by mtRNase P. Multiple-turnover kinetic analyses of pre-tRNA cleavage were performed with 1 nM PRORP in the presence of an excess of TRMT10C-SDR5C1 complex and with varying concentrations of pre-tRNA (at 4.5 mM Mg²⁺). Initial velocities of cleavage (v; see Materials and Methods, and Supplementary Figure S5) were plotted against the pre-tRNA concentration. Data points are the mean ± SEM of at least five replicates. Derived kinetic constants k_cat and K_M (best-fit values ± curve-fit standard error) are inserted into each graph. (A, B) Multiple-turnover kinetic analysis of the cleavage of (A) pre-tRNA^Met and (B) pre-tRNA^Lys by mtRNase P.

Figure 8.

Pulse-chase kinetics of pre-tRNA cleavage by mtRNase P. Trace amounts of pre-tRNA were cleaved by an excess of mtRNase P (PRORP + TRMT10C-SDR5C1) at 4.5 mM Mg²⁺, and the reactions were quenched (‘chase’) after 20 s, either by addition of a 1000-fold excess of unlabeled substrate, or by 200-fold dilution with reaction buffer. Aliquots were withdrawn and the reaction stopped at the indicated time points. Samples were analyzed by gel electrophoresis and phosphorimaging, and the cleaved fraction of pre-tRNA was determined by image analysis. Control reactions without a ‘chase’ and with the ‘chase’ prior to the start of the reaction (t₀) were run in parallel. Pulse-chase kinetic analysis of (A) pre-tRNA^Met and of (B) pre-tRNA^Lys cleavage by mtRNase P.