Nuclear Protein-Only Ribonuclease P2 Structure and Biochemical Characterization Provide Insight into the Conserved Properties of tRNA 5′ End Processing Enzymes

Abstract

Protein-only RNase Ps (PRORPs) are a recently discovered class of RNA processing enzymes that catalyze maturation of the 5′ end of precursor tRNAs (pre-tRNAs) in Eukaryotes. PRORPs are found in the nucleus and/or organelles of most eukaryotic organisms. Arabidopsis thaliana is a representative organism that contains PRORP enzymes (PRORP1, PRORP2, PRORP3) in both its nucleus and organelles; PRORP2 and 3 localize to the nucleus, PRORP1 to the chloroplast and the mitochondria. Apart from their identification, almost nothing is known about the structure and function of PRORPs that act in the nucleus. Here we use a combination of biochemical assays and X-ray crystallography to characterize A. thaliana PRORP2. We solved the crystal structure of PRORP2 (3.2 Å) revealing an overall V-shaped protein and conserved metallonuclease active site structure. Our biochemical studies indicate that PRORP2 requires Mg2+ for catalysis and catalyzes the maturation of nuclear encoded substrates up to 10-fold faster than mitochondrial encoded precursor nad6 t-element under single turnover conditions. We also demonstrate that PRORP2 preferentially binds pre-tRNAs containing short 5′ leaders and 3′ trailers; however, leader and trailer length do not significantly alter the observed rate constants of PRORP2 in single turnover cleavage assays. Our data provide a biochemical and structural framework to begin understanding how nuclear localized PRORPs recognize and cleave their substrates.

Differential substrate recognition by isozymes of plant protein-only Ribonuclease P

“…Thus, PRORP enzymes do not share a major determinant of molecular recognition utilized by bacterial RNase P. Furthermore, PRORP1 and B. subtilis RNase P bind mature tRNA ∼30-and 400-fold weaker than pre-tRNA (leader length ≥ 4 nts), respectively . This is consistent with PRORP1 making fewer interactions with the leader than B. subtilis RNase P. Similar to PRORP1, recent binding studies with A. thaliana nuclear pre-tRNA Gly suggest PRORP2 does not require long leader (≤8 nt) and trailer (1 nt) lengths for tight binding (Karasik et al 2016). PRORPs efficiently catalyze removal of leader sequences from CysMito tRNA lacking an anti-codon stem-loop (ΔAC) but not from a model substrate lacking the anticodon stemloop and D-arm (SL substrate).…”

“…However, full-length PRORP2 expressed in E. coli is observed mainly in the soluble fraction. A Δ20 amino acid truncation of PRORP2 at the N-terminus (the comparable truncation to Δ76 PRORP1) has comparable activity to full-length PRORP2 (Karasik et al 2016).…”

Interplay between substrate recognition, 5’ end tRNA processing and methylation activity of human mitochondrial RNase P

“…In non-metazoan eukaryotes, the MRPP3-homologous PRORP enzymes are stand-alone proteins able to cleave a wide variety of substrates from different organisms (Gobert et al 2010;Karasik et al 2016;Howard, Klemm, and Fierke 2015). To gain insight into potential differences as to how single-protein PRORPs and complex-forming MRPP3 enzymes select and cleave their substrates we assessed the ability of A. thaliana PRORP1 and PRORP2 to recognize and process human (mt)pre-tRNAs.…”

“…Initial steps for protein expression and purification, including nickel column purification, histidine tag and TEV removal are identical to those detailed above for Δ95MRPP3, with the following exceptions: the antibiotic used was streptomycin (50 µg/ml) and 10% glycerol was added to each Therefore, protein concentration was measured based on molecular weight and extinction coefficient values of 149 kDa and 79520 cm -1 mol -1 . A. thaliana PRORP1 and PRORP2 were expressed and purified from E. coli as previously described (Howard et al 2012;Karasik et al 2016). MRPP3 in cleavage reaction buffer were added to 20 nM fluorescently labeled pre-tRNA.…”

“…In contrast to human mtRNase P, PRORPs are stand-alone enzymes that do not require accessory proteins. Most studies of protein-based RNase Ps have focused on PRORPs because these enzymes are homologous to MRPP3 and provide a tractable system for study (Pinker et al 2017;Gobert et al 2013;Gutmann, Gobert, and Giege 2012;Gobert et al 2010;Karasik et al 2016;Howard et al 2013;Howard et al 2012;Howard et al 2016;Klemm et al 2016). As a consequence, little is known about how the components of the metazoan mtRNase P complex function.…”