Pentatricopeptide repeat motifs in the processing enzyme PRORP1 in Arabidopsis thaliana play a crucial role in recognition of nucleotide bases at TψC loop in precursor tRNAs

Imai, Takayoshi; Nakamura, Takahiro; Maeda, Taku; Nakayama, Kaoru; Gao, Xuzhu; Nakashima, Takashi; Kakuta, Yoshimitsu; Kimura, Makoto

doi:10.1016/j.bbrc.2014.07.030

Cited by 25 publications

(40 citation statements)

References 30 publications

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“…Since we could map neither the first 28 residues of PRORP2 nor the residues between the localization signal and the 94 th residue in PRORP1, we predict that these regions may be structurally disordered. It was previously suggested that this region might be functionally important based on reduced activity of truncated versions of PRORP1 14 . To test if this region is indeed important for the catalytic activity of PRORP2, we measured the single-turnover rate constants for PRORP2 containing truncations at the N- tand C-termini (residues 21–515).…”

Section: Resultsmentioning

confidence: 99%

“…It has been proposed that PPR domains interact with RNAs via both the RNA sugar-phosphate backbone and/or base specific contacts 14; 29; 49 . These types of contacts are more likely to be made between the PRORP PPR domains and pre-tRNA D- and T-arms, and the leader sequences 49 than with pre-tRNA aniticodon loops.…”

Section: Discussionmentioning

confidence: 99%

“…The Nedd4-BP1, YacP nuclease (NYN) domain (metallonuclease domain) houses the active site that catalyzes 5′ pre-tRNA cleavage. The functional importance of this domain has been validated in mutagenesis studies where mutations of conserved aspartates to alanines resulted in catalytically inactive PRORP1 8; 14 . The central domain holds the PPR and the metallonuclease domain together in such a way that the two functionally relevant domains face towards each other.…”

Section: Introductionmentioning

confidence: 99%

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

Karasik

Shanmuganathan

Howard

et al. 2016

Journal of Molecular Biology

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Karasik

Shanmuganathan

Howard

et al. 2016

Journal of Molecular Biology

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“…The PPR domain contains five PPR or PPR-like motifs, which are helix-turn-helix motifs found in tandem. This domain has been proposed to interact with pre-tRNA and enhance binding affinity (Howard et al 2012;Gobert et al 2013;Imai et al 2014). The largest sequence variation among the PRORPs is found in the PPR domains and thus differences in substrate recognition may lie within this region.…”

Section: Introductionmentioning

confidence: 99%

“…Previous studies have identified the importance of the elbow region of tRNA for PRORP1 substrate recognition (Gobert et al 2013;Imai et al 2014). However, recognition of the tRNA leader and trailer by PRORP is largely unknown.…”

Section: Introductionmentioning

confidence: 99%

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

Howard

Karasik

Klemm

et al. 2016

RNA

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Ribonuclease P (RNase P) catalyzes the cleavage of leader sequences from precursor tRNA (pre-tRNA). Typically, these enzymes are ribonucleic protein complexes that are found in all domains of life. However, a new class of RNase P has been discovered that is composed entirely of protein, termed protein-only RNase P (PRORP). To investigate the molecular determinants of PRORP substrate recognition, we measured the binding affinities and cleavage kinetics of Arabidopsis PRORP1 for varied pre-tRNA substrates. This analysis revealed that PRORP1 does not make significant contacts within the trailer or beyond N −1 of the leader, indicating that this enzyme recognizes primarily the tRNA body. To determine the extent to which sequence variation within the tRNA body modulates substrate selectivity and to provide insight into the evolution and function of PRORP enzymes, we measured the reactivity of the three Arabidopsis PRORP isozymes (PRORP1-3) with four pre-tRNA substrates. A 13-fold range in catalytic efficiencies (10 4 -10 5 M −1 s −1 ) was observed, demonstrating moderate selectivity for pre-tRNA substrates. Although PRORPs bind the different pre-tRNA species with affinities varying by as much as 100-fold, the three isozymes have similar affinities for a given pre-tRNA, suggesting similar binding modes. However, PRORP isozymes have varying degrees of cleavage fidelity, which is dependent on the pre-tRNA species and the presence of a 3 ′ -discriminator base. This work defines molecular determinants of PRORP substrate recognition that provides insight into this new class of RNA processing enzymes.

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Recent Progress Toward RNA Manipulation with Engineered Pentatricopeptide Repeat Proteins

Imai

Yagi

Nakamura

2018

Applied RNA Bioscience

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Pentatricopeptide repeat motifs in the processing enzyme PRORP1 in Arabidopsis thaliana play a crucial role in recognition of nucleotide bases at TψC loop in precursor tRNAs

Cited by 25 publications

References 30 publications

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

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

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

Recent Progress Toward RNA Manipulation with Engineered Pentatricopeptide Repeat Proteins

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