Cleavage of Model Substrates by Arabidopsis thaliana PRORP1 Reveals New Insights into Its Substrate Requirements

Abstract

Two broad classes of RNase P trim the 5' leader of precursor tRNAs (pre-tRNAs): ribonucleoprotein (RNP)- and proteinaceous (PRORP)-variants. These two RNase P types, which use different scaffolds for catalysis, reflect independent evolutionary paths. While the catalytic RNA-based RNP form is present in all three domains of life, the PRORP family is restricted to eukaryotes. To obtain insights on substrate recognition by PRORPs, we examined the 5' processing ability of recombinant Arabidopsis thaliana PRORP1 (AtPRORP1) using a panel of pre-tRNASer variants and model hairpin-loop derivatives (pATSer type) that consist of the acceptor-T-stem stack and the T-/D-loop. Our data indicate the importance of the identity of N-1 (the residue immediately 5' to the cleavage site) and the N-1:N+73 base pair for cleavage rate and site selection of pre-tRNASer and pATSer. The nucleobase preferences that we observed mirror the frequency of occurrence in the complete suite of organellar pre-tRNAs in eight algae/plants that we analyzed. The importance of the T-/D-loop in pre-tRNASer for tight binding to AtPRORP1 is indicated by the 200-fold weaker binding of pATSer compared to pre-tRNASer, while the essentiality of the T-loop for cleavage is reflected by the near-complete loss of activity when a GAAA-tetraloop replaced the T-loop in pATSer. Substituting the 2'-OH at N-1 with 2'-H also resulted in no detectable cleavage, hinting at the possible role of this 2'-OH in coordinating Mg2+ ions critical for catalysis. Collectively, our results indicate similarities but also key differences in substrate recognition by the bacterial RNase P RNP and AtPRORP1: while both forms exploit the acceptor-T-stem stack and the elbow region in the pre-tRNA, the RNP form appears to require more recognition determinants for cleavage-site selection.

Fig 1. Secondary structures of substrates used in this study.

Secondary structures of pSu1 and pATSer. The highlighted regions/residues were substituted to generate the different variants as indicated, A, adenosine, G, guanosine, U, uridine, I (Ino), inosine and D (DAP), 2,6-diaminopurine; dC, deoxycytosine; and dU, deoxyuridine. The canonical RNase P cleavage sites between residues N_-1 and N₊₁ (correct cleavage denoted C₀), and the alternative cleavage sites between residues N_-2 and N_-1 (miscleavage denoted as M_-1) are marked with black and grey arrows, respectively. The N₊₇₃ position, which immediately precedes the 3'-terminal CCA-motif, corresponds to the discriminator base.

Fig 2. AtPRORP1-mediated cleavage of pre-tRNA^SerSu1 (pSu1).

Representative gel showing AtPRORP1-mediated cleavage of pre-tRNA^SerSu1 (pSu1) substrates with and without the 3' CCA. Lanes 1 to 8 represent negative controls (absence of AtPRORP1), and M (size marker, lane 9) indicates cleavage of pATSerUG by Eco RPR. Note that this cleavage generates a 5' cleavage fragment (5' CL Frags) one nucleotide longer compared to that generated during cleavage of pSu1. Lanes 10 and 14 pSu1(-1A), lanes 11 and 15 pSu1(-1C), lanes 12 and 16 pSu1(-1G), and lanes 13 and 17 pSu1(-1U). The final concentration of AtPRORP1 was 0.37 μM and the reactions were performed at 37°C for 30 s in the presence of 10 mM Mg²⁺ (see Materials and Methods).

Fig 3. Frequencies of cleavage-site selection by AtPRORP1.

Histograms summarizing cleavage-site selection frequencies (in %) during AtPRORP1-mediated cleavage of pSu1 "-1" (A) and pATSer (B) variants. Mean and standard deviation values were calculated using data from at least three independent experiments.

Fig 4. Effect of varying Mg²⁺ concentration on AtPRORP1-mediated cleavage.

AtPRORP1-mediated cleavage of the pSu1(-1U) (A) and pATSerUG (B) as a function of Mg²⁺ concentration at 37°C. Mean and standard deviation values were calculated using data from at least three independent experiments.

Fig 5. AtPRORP1-mediated cleavage of pATSer variants.

Representative gel showing AtPRORP1-mediated cleavage of 3' CCA-motif-containing pATSer variants. Lanes 1 to 6 and 14 to 17 are negative controls (loaded in the same order as the reactions with AtPRORP1 in lanes 8 to 13 and 19 to 22, respectively); and lanes 7 and 18 (size marker) refers to cleavage of pATSerUG by Eco RPR. The final concentration of AtPRORP1 was 6.6 μM and the reactions were performed at 37°C for 60 min in the presence of 10 mM Mg²⁺. The position of each 5' cleavage fragment (5' CL Frags) generated after cleavage is indicated. The two lower panels represent overexposure to better highlight the 5'-cleavage products in the upper panels. (Note: Fig A in S1 File shows cleavage of pATSer derivatives without the 3' CCA-motif.)

Fig 6. Analysis of N_-1:N₊₇₃ identities in mitochondrial and chloroplast tRNAs.

Analysis of N_-1:N₊₇₃ identities in 423 mitochondrial and chloroplast tRNAs from eight different green algae and plants (sequences obtained from http://plantrna.ibmp.cnrs.fr/). Table A in S1 file lists the individual distributions in each species.