PPR proteins shed a new light on RNase P biology

Abstract

A fast growing number of studies identify pentatricopeptide repeat (PPR) proteins as major players in gene expression processes. Among them, a subset of PPR proteins called PRORP possesses RNase P activity in several eukaryotes, both in nuclei and organelles. RNase P is the endonucleolytic activity that removes 5' leader sequences from tRNA precursors and is thus essential for translation. Before the characterization of PRORP, RNase P enzymes were thought to occur universally as ribonucleoproteins, although some evidence implied that some eukaryotes or cellular compartments did not use RNA for RNase P activity. The characterization of PRORP reveals a two-domain enzyme, with an N-terminal domain containing multiple PPR motifs and assumed to achieve target specificity and a C-terminal domain holding catalytic activity. The nature of PRORP interactions with tRNAs suggests that ribonucleoprotein and protein-only RNase P enzymes share a similar substrate binding process.

Keywords: RNA binding protein; RNase P; evolution; pentatricopeptide repeat; tRNA maturation.

Figure 1. Plant RNase Ps do not contain an RNA component. (A) Resistance of spinach chloroplast RNase P to digestion with micrococcal nuclease (MN). Crude enzyme fraction was incubated with the indicated amounts of MN plus 5 mM CaCl₂ (30 min at 37°C) after which excess EGTA was added, followed by substrate and reaction buffer. Lane 1, positive control for RNase P (pre-incubated without MN); lane 2, positive control for MN (as lane 1 but MN not inactivated prior to addition of substrate); lanes 3–6, pre-incubated with 2‒40 U MN/μl and treated with EGTA prior to assay; lanes 7–10, as lanes 3–6 with addition of 1 μg poly(A)/μl prior to assay. Modified from reference . (Essentially identical results were obtained with wheat nuclear RNase P.⁴⁸) (B) Buoyant density of spinach chloroplast RNase P. Fraction II chloroplast enzyme (~5 mg) was pretreated with MN (1 U/μl, 20 min; terminated with EGTA), brought up to 1.0 ml with gradient buffer, and layered over 4.0 ml of CsCl solution (1.40 g/ml). After centrifugation to equilibrium, fractions were collected from the top and density was determined by refractometry. CsCl was removed by dialysis and fractions were assayed for RNase P. Lower panel, distribution across the gradient of total protein (filled squares) and RNase P activity (open circles: amol mature tRNA formed; shaded circles: non-tRNA-sized material). Upper panel, observed buoyant density of each fraction.

Figure 2. The occurrence of PRORP in eukaryote lineages is represented in an unrooted neighbor-joining phylogenic tree derived from Gobert et al. Representative PRORP protein sequences described by Gobert et al. from evolutionarily distant eukaryotes were used for the phylogenetic analysis. Grey names show the incidence of putative PRORP sequences in the respective subgroups whereas black names indicate species where PRORP proteins were experimentally shown to hold RNase P activity. The demonstration that RNase P activity could be held by PRORP proteins in distantly related eukaryote groups such as Metazoa, Euglenozoa and Viridiplantae strongly suggest that PRORP evolved early in eukaryote history.

Figure 3. PRORP are two-domain PPR proteins. (A) 3D models were built using SwissModel for all characterized members of the PRORP family based on At-PRORP1 crystal structure (PDB ID 4G24). This global view shows superimposed structure and models with PPR domains in blue, N-terminal and C-terminal connecting regions in orange and yellow, respectively, and catalytic NYN domains in green. Insertions/deletions to the reference structure of At-PRORP1 are colored as following: A. thaliana PRORP2, PRORP3, O. tauri PRORP, Trypanosoma PRORP2 and human mitochondrial MRPP3 indels are in violet, red, dark green, light brown and pink, respectively. Little structural variations are observed. (B) At-PRORP1 PPR domain. (Left) This view of the whole domain highlights individual PPR motifs in light to dark blue from N to C terminus. (Right) Superposition of the five PPR motifs from A. thaliana PRORP1 (represented with the same color code as on the left) and the two PPR motifs (in orange and yellow) of human mitochondrial RNA polymerase (PDB ID 3SPA) illustrating the conservation of the PPR fold. (C) At-PRORP1 catalytic domain. Manganese ions shown as pink spheres and two water molecules bridging one Mn²⁺ ion to conserved Asp474 in the catalytic site. (D) At-PRORP1 connecting region. This region is composed of a N-terminal half (orange) following the PPR domain and a C-terminal half (yellow) following the catalytic domain. It binds a zinc ion (gray) coordinated by C344, C345 (orange) and H548, C565 (yellow).

Figure 4. The current model of the PRORP/tRNA complex suggests a common mode of RNA binding in RNP and PRORP RNases P. (A) Structure of Thermotoga maritima ribozyme (PDBid 3Q1R18) with the catalytic domain in green, the specificity domain in blue, the RNase P protein subunit in orange, the tRNA product in light blue and the molecular surface of the RNP in gray. (B) The two-domain architecture of At-PRORP1 structure offers a concave surface that can be docked on the tRNA acceptor arm. The protein shown in the same orientation and same color code as the RNP with the catalytic domain in green, with metal ions bound (yellow spheres) close to the RNA cleavage site and the RNA-binding PPR domain in blue interacting with the region of the D-TψC loops. The central region (yellow) stabilized by a zinc ion (orange sphere) connects the two main PRORP domains. (C) A close-up of the PRORP1-tRNA complex model shows conserved catalytic aspartates D474 and D475 (blue) adjacent to tRNA cleavage site at position G+1 (red) as well as U16, G18, G19 and C56 (the nucleotides protected in footprint experiments in red) in contact with PPR motifs. Current functional data indicate that PRORP proteins have evolved an RNA recognition process very similar to that of RNP RNase P.