Pre-tRNA turnover catalyzed by the yeast nuclear RNase P holoenzyme is limited by product release

Hsieh, John; Walker, Scott C.; Fierke, Carol A.; Engelke, David R.

doi:10.1261/rna.1309409

Cited by 28 publications

(41 citation statements)

References 60 publications

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“…1B). These parameters are comparable with those observed for eukaryotic ribonucleoprotein RNase P (Hsieh et al 2009). Altogether, with these results, we found that PRORP proteins whose localization is strictly restricted to nuclei in Arabidopsis have canonical RNase P activity as single-protein enzymes.…”

Section: Resultssupporting

confidence: 85%

PRORP proteins support RNase P activity in both organelles and the nucleus in Arabidopsis

Gutmann¹,

Gobert²,

Giegé³

2012

Genes Dev.

120

167

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RNase P is an essential enzyme that cleaves the 59 leader sequence of tRNA precursors. RNase Ps were believed until now to occur universally as ribonucleoproteins in organisms performing RNase P activity. Here we find that protein-only RNase P enzymes called PRORP (for proteinaceous RNase P) support RNase P activity in vivo in both organelles and the nucleus in Arabidopsis. Beyond tRNA, PRORP proteins are involved in the maturation of small nucleolar RNA (snoRNA) and mRNA. Finally, ribonucleoprotein RNase MRP is not involved in tRNA maturation in plants. Altogether, our results indicate that ribonucleoprotein enzymes have been entirely replaced by proteins for RNase P activity in plants.Supplemental material is available for this article.Received February 10, 2012; revised version accepted April 5, 2012.RNase P is a virtually universal enzyme involved in the maturation of tRNAs, as it cleaves the 59 leader sequence of tRNA precursors. It is thus essential to obtain functional tRNAs and is therefore pivotal for translation (Lai et al. 2010;Reiter et al. 2010). RNase P activities from all phyla of life were assumed to be universally performed by ribonucleoprotein enzymes whose catalytic activities are held by ribozymes (Altman 2007). This concept was first challenged with the proposition that spinach chloroplast and human mitochondria RNase Ps would not contain any RNA moiety (Wang et al. 1988;Rossmanith and Karwan 1998). More recently, protein-only RNase P enzymes called PRORP (for proteinaceous RNase P) have been characterized at the molecular level in endosymbiotic organelles in both humans and Arabidopsis (Holzmann et al. 2008;Gobert et al. 2010). Still, the dogma remained that RNase P enzymes would nonetheless universally occur as ribonucleoproteins in living organisms performing RNase P activity, with protein-only RNase Ps being marginal exceptions restricted to only some organelles (e.g., Esakova and Krasilnikov 2010).The putative PRORP RNase P enzymes are characterized by the occurrence of a conserved ''NYN'' metallonuclease domain (Anantharaman and Aravind 2006). PRORP proteins also belong to the huge pentatricopeptide repeat (PPR) protein family. These proteins, typically from eukaryotes, are involved in a wide variety of posttranscriptional mechanisms (Schmitz-Linneweber and Small 2008). However, no functional information was available for PRORP proteins. We previously established that Arabidopsis PRORP1, a protein localized in organelles, can act in vitro as an RNase P enzyme that is a single protein (Gobert et al. 2010), although its function remained elusive in planta. We also used localization experiments (YFP fusions and immunodetections) to determine that PRORP2 and PRORP3, two paralogs of PRORP1, were present in Arabidopsis nuclei (Gobert et al. 2010). In addition, the fast-growing amount of genomic data has revealed that some important groups of eukaryotes, such as land plants and kinetoplastids, do not encode any recognizable genes for RNase P RNA or for proteins specific for ribonucleoprotein...

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Section: Resultssupporting

confidence: 85%

PRORP proteins support RNase P activity in both organelles and the nucleus in Arabidopsis

Gutmann¹,

Gobert²,

Giegé³

2012

Genes Dev.

120

167

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“…1C, inset). The observation of burst kinetics indicated that the rate-limiting step of the reaction is product release (Hartley and Kilby 1954), as reported for a number of other nuclease ribozymes (Herschlag and Cech 1990a,b;Young et al 1991;Stage-Zimmermann and Uhlenbeck 1998;Shih and Been 2000;Hsieh et al 2009). The apparent k cat during the presteady state (k burst = 0.36 ± 0.04 min −1 ), calculated from the burst phase, was close to that previously measured with radiolabeled substrates under single-turnover conditions (k obs = 0.2 min −1 ) (Lazarev et al 2003) but the turnover number in the steady state (k ss cat ), was ∼20-fold lower (Table 1).…”

Section: X-motif Activity Under Multiple-turnover Conditionssupporting

confidence: 72%

“…1C), indicating that product release is no longer rate limiting. These observations, together with the location of most of the selected positive mutations in substrate-binding site of iXms ribozymes, indicate that the elevated k ss cat of the iXms results mainly from a reduction of affinity for the reaction products which allows more rapid product release and turnover, but that the changes required to decrease product affinity result in an even more dramatic decrease in the affinity for the substrate, as proposed for other ribozymes (Herschlag and Cech 1990a,b;Young et al 1991;StageZimmermann and Uhlenbeck 1998;Shih and Been 2000;Hsieh et al 2009). …”

Section: Characterization Of Selected Variantsmentioning

confidence: 76%

Using droplet-based microfluidics to improve the catalytic properties of RNA under multiple-turnover conditions

Ryckelynck

Baudrey

Rick

et al. 2015

RNA

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In vitro evolution methodologies are powerful approaches to identify RNA with new functionalities. While Systematic Evolution of Ligands by Exponential enrichment (SELEX) is an efficient approach to generate new RNA aptamers, it is less suited for the isolation of efficient ribozymes as it does not select directly for the catalysis. In vitro compartmentalization (IVC) in aqueous droplets in emulsions allows catalytic RNAs to be selected under multiple-turnover conditions but suffers severe limitations that can be overcome using the droplet-based microfluidics workflow described in this paper. Using microfluidics, millions of genes in a library can be individually compartmentalized in highly monodisperse aqueous droplets and serial operations performed on them. This allows the different steps of the evolution process (gene amplification, transcription, and phenotypic assay) to be uncoupled, making the method highly flexible, applicable to the selection and evolution of a variety of RNAs, and easily adaptable for evolution of DNA or proteins. To demonstrate the method, we performed cycles of random mutagenesis and selection to evolve the X-motif, a ribozyme which, like many ribozymes selected using SELEX, has limited multiple-turnover activity. This led to the selection of variants, likely to be the optimal ribozymes that can be generated using point mutagenesis alone, with a turnover number under multiple-turnover conditions, k ss cat , ∼28-fold higher than the original X-motif, primarily due to an increase in the rate of product release, the rate-limiting step in the multiple-turnover reaction.

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“…However, the efficiency [estimated from k cat /K M for PRORP3 (∼6 × 10 4 M −1 ·s −1 ) (3) and k chem /K 1/2 for PRORP1 (∼5 × 10 4 M −1 ·s −1 )] for catalysis of pre-tRNA cleavage under in vitro conditions is slower than that for catalysis by Bacillus subtilis (∼100-fold) or Saccharomyces cerevisiae (∼30-fold) RNase P under similar conditions (3,36,37). Nonetheless, PRORPs can complement Escherichia coli RNase P and the large multicomponent yeast nuclear RNase P in vivo (2,38).…”

Section: Discussionmentioning

confidence: 99%

Mitochondrial ribonuclease P structure provides insight into the evolution of catalytic strategies for precursor-tRNA 5′ processing

Howard¹,

Lim²,

Fierke

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

109

235

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Ribonuclease P (RNase P) catalyzes the maturation of the 5′ end of tRNA precursors. Typically these enzymes are ribonucleoproteins with a conserved RNA component responsible for catalysis. However, protein-only RNase P (PRORP) enzymes process precursor tRNAs in human mitochondria and in all tRNA-using compartments of Arabidopsis thaliana. PRORP enzymes are nuclear encoded and conserved among many eukaryotes, having evolved recently as yeast mitochondrial genomes encode an RNase P RNA. Here we report the crystal structure of PRORP1 from A. thaliana at 1.75 Å resolution, revealing a prototypical metallonuclease domain tethered to a pentatricopeptide repeat (PPR) domain by a structural zinc-binding domain. The metallonuclease domain is a unique high-resolution structure of a Nedd4-BP1, YacP Nucleases (NYN) domain that is a member of the PIN domain-like fold superfamily, including the FLAP nuclease family. The structural similarity between PRORP1 and the FLAP nuclease family suggests that they evolved from a common ancestor. Biochemical data reveal that conserved aspartate residues in PRORP1 are important for catalytic activity and metal binding and that the PPR domain also enhances activity, likely through an interaction with pre-tRNA. These results provide a foundation for understanding tRNA maturation in organelles. Furthermore, these studies allow for a molecular-level comparison of the catalytic strategies used by the only known naturally evolved protein and RNA-based catalysts that perform the same biological function, pre-tRNA maturation, thereby providing insight into the differences between the prebiotic RNA world and the present protein-dominated world.catalytic mechanism | magnesium | molecular recognition A ccording to the RNA world hypothesis, RNA played dual roles as carrier of genetic information and catalyst in a prebiotic world. However, over eons of evolution, proteins with their expanded 20-aa alphabet and greater structural and functional complexity took over many RNA-based functions. Structural insights into this evolutionary transition are limited because of the lack of examples of RNA and protein macromolecules that perform the same biological function in nature. One notable exception is ribonuclease P (RNase P) that catalyzes maturation of the 5′ end of tRNA across all domains of life. Until recently, all known RNase P enzymes included a catalytic RNA component. The discovery of a protein-only RNase P [proteinacous RNase P (PRORP)] from human mitochondria and Arabidopsis thaliana has dramatically shifted this paradigm (1-3). These enzymes represent a unique class of metallonucleases and are conserved among many eukaroytes (1, 2). A. thaliana encodes three PRORP enzymes (PRORP1, -2, and -3) that catalyze pretRNA processing (2). PRORP1 localizes to the mitochondria and chloroplast whereas PRORP2 and PRORP3 localize to the nucleus (2), suggesting that protein-based enzymes catalyze pretRNA maturation in these cellular locations (2, 3). To gain mechanistic and evolutionary insights into the PRORP...

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Pre-tRNA turnover catalyzed by the yeast nuclear RNase P holoenzyme is limited by product release

Cited by 28 publications

References 60 publications

PRORP proteins support RNase P activity in both organelles and the nucleus in Arabidopsis

PRORP proteins support RNase P activity in both organelles and the nucleus in Arabidopsis

Using droplet-based microfluidics to improve the catalytic properties of RNA under multiple-turnover conditions

Mitochondrial ribonuclease P structure provides insight into the evolution of catalytic strategies for precursor-tRNA 5′ processing

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