Ribonuclease P: the evolution of an ancient RNA enzyme

Ribonuclease P (RNase P) is an ancient and essential endonuclease that catalyses the cleavage of the 5' leader sequence from precursor tRNAs (pre-tRNAs). The enzyme is one of only two ribozymes which can be found in all kingdoms of life (Bacteria, Archaea, and Eukarya). Most forms of RNase P are ribonucleoproteins; the bacterial enzyme possesses a single catalytic RNA and one small protein. However, in archaea and eukarya the enzyme has evolved an increasingly more complex protein composition, whilst retaining a structurally related RNA subunit. The reasons for this additional complexity are not currently understood. Furthermore, the eukaryotic RNase P has evolved into several different enzymes including a nuclear activity, organellar activities, and the evolution of a distinct but closely related enzyme, RNase MRP, which has different substrate specificities, primarily involved in ribosomal RNA biogenesis. Here we examine the relationship between the bacterial and archaeal RNase P with the eukaryotic enzyme, and summarize recent progress in characterizing the archaeal enzyme. We review current information regarding the nuclear RNase P and RNase MRP enzymes in the eukaryotes, focusing on the relationship between these enzymes by examining their composition, structure and functions.     

An active precursor in assembly of yeast nuclear ribonuclease P

The RNA-protein subunit assembly of nuclear RNase P was investigated by specific isolation and characterization of the precursor and mature forms of RNase P using an RNA affinity ligand. Pre-RNase P was as active in pre-tRNA cleavage as mature RNase P, although it contained only seven of the nine proteins found in mature RNase P. Pop3p and Rpr2p were not required for maturation of the RPR1 RNA subunit and virtually absent from pre-RNase P, implying that they are dispensable for pre-tRNA substrate recognition and cleavage. The RNase P subunit assembly is likely to occur in the nucleolus, where both precursor and mature forms of RNase P RNA are primarily localized. The results provide insight into assembly of nuclear RNase P, and suggest pre-tRNA substrate recognition is largely determined by the RNA subunit.     

Binding and cleavage of unstructured RNA by nuclear RNase P

Ribonuclease P (RNase P) is an essential endoribonuclease for which the best-characterized function is processing the 5' leader of pre-tRNAs. Compared to bacterial RNase P, which contains a single small protein subunit and a large catalytic RNA subunit, eukaryotic nuclear RNase P is more complex, containing nine proteins and an RNA subunit in Saccharomyces cerevisiae. Consistent with this, nuclear RNase P has been shown to possess unique RNA binding capabilities. To understand the unique molecular recognition of nuclear RNase P, the interaction of S. cerevisiae RNase P with single-stranded RNA was characterized. Unstructured, single-stranded RNA inhibits RNase P in a size-dependent manner, suggesting that multiple interactions are required for high affinity binding. Mixed-sequence RNAs from protein-coding regions also bind strongly to the RNase P holoenzyme. However, in contrast to poly(U) homopolymer RNA that is not cleaved, a variety of mixed-sequence RNAs have multiple preferential cleavage sites that do not correspond to identifiable consensus structures or sequences. In addition, pre-tRNA(Tyr), poly(U)(50) RNA, and mixed-sequence RNA cross-link with purified RNase P in the RNA subunit Rpr1 near the active site in "Conserved Region I," although the exact positions vary. Additional contacts between poly(U)(50) and the RNase P proteins Rpr2p and Pop4p were identified. We conclude that unstructured RNAs interact with multiple protein and RNA contacts near the RNase P RNA active site, but that cleavage depends on the nature of interaction with the active site.     

Ribonuclease P from plant nuclei and photosynthetic organelles

RNase P consists of both protein and RNA subunits in all organisms and organelles investigated so far, with the exception of chloroplasts and plant nuclei where no enzyme-associated RNA has been detected to date. Studies on substrate specificity revealed that cleavage by plant nuclear RNase P is critically dependent on a complete and intact structure of the substrate. No clearcut answer is yet possible regarding the order of processing events at the 5 or 3 end of tRNAs in the case of nuclear or chloroplast processing enzymes. RNase P from a phylogenetically ancient photosynthetic organelle will be discussed in greater detail: The enzyme from theCyanophora paradoxa cyanelle is the first RNase P from a photosynthetic organelle which has been shown to contain an essential RNA subunit. This RNA is strikingly similar to its counterpart from cyanobacteria, yet it lacks catalytic activity. Properties of the holoenzyme suggest an intermediate position in RNA enzyme evolution, with an eukaryotic-type, inactive RNA and a prokaryotic-type small protein subunit. The possible presence of an RNA component in RNase P from plant nuclei and modern chloroplasts will be discussed, including a critical evaluation of some criteria that have been frequently applied to elucidate the subunit composition of RNase P from different organisms.Abbreviations RNase P Ribonuclease P - (pre-)tRNA transfer ribonucleic acid (precursor) - tRNA ^Ser (- ^Tyr , - ^Phe ) transfer ribonucleic acid specific for serine (tyrosine, phenylalanine) - CyRP RNA RNA component of cyanelle RNase P     

Structure-function analysis in nuclear RNase P RNA

Eukaryotic ribonuclease P (RNase P) enzymes require both RNA and protein subunits for activityin vivo andin vitro. We have undertaken an analysis of the complex RNA subunit of the nuclear holoenzyme in an effort to understand its structure and its similarities to and differences from the bacterial ribozymes. Phylogenetic analysis, structure-sensitive RNA footprinting, and directed mutagenesis reveal conserved secondary and tertiary structures with both strong similarities to the bacterial consensus and distinctive features. The effects of mutations in the most highly conserved positions are being used to dissect the functions of individual subdomains.Abbreviations RPRI ribonucleaseP ribonucleoprotein 1 gene fromSaccharomyces cerevisiae - Pu purine ribonucleoside     

Evidence for an RNA-based catalytic mechanism in eukaryotic nuclear ribonuclease P

Ribonuclease P is the enzyme responsible for removing the 5'-leader segment of precursor transfer RNAs in all organisms. All eukaryotic nuclear RNase Ps are ribonucleoproteins in which multiple protein components and a single RNA species are required for activity in vitro as well as in vivo. It is not known, however, which subunits participate directly in phosphodiester-bond hydrolysis. The RNA subunit of nuclear RNase P is evolutionarily related to its catalytically active bacterial counterpart, prompting speculation that in eukaryotes the RNA may be the catalytic component. In the bacterial RNase P reaction, Mg(II) is required to coordinate the nonbridging phosphodiester oxygen(s) of the scissile bond. As a consequence, bacterial RNase P cannot cleave pre-tRNA in which the pro-Rp nonbridging oxygen of the scissile bond is replaced by sulfur. In contrast, the RNase P reaction in plant chloroplasts is catalyzed by a protein enzyme whose mechanism does not involve Mg(II) coordinated by the pro-Rp oxygen. To determine whether the mechanism of nuclear RNase P resembles more closely an RNA- or a protein-catalyzed reaction, we analyzed the ability of Saccharomyces cerevisiae nuclear RNase P to cleave pre-tRNA containing a sulfur substitution of the pro-Rp oxygen at the cleavage site. Sulfur substitution at this position prohibits correct cleavage of pre-tRNA. Cleavage by eukaryotic RNase P thus depends on the presence of a thio-sensitive ligand to the pro-Rp oxygen of the scissile bond, and is consistent with a common, RNA-based mechanism for the bacterial and eukaryal enzymes.     

DNA-dependent RNA polymerases isolated from yeast mitochondria

Purified preparations of yeast mitochondria yield three species of DNA-dependent RNA polymerases. These enzymes have been separated and purified to homogeneity for analysis of their properties and for comparison with the properties of nuclear preparations of yeast RNA polymerases. Three enzymes have been separated by DEAE-Sephadex chromatography of each fraction. Both nuclear and mitochondrial preparations yield three components with nearly identical elution properties. The distributions of enzyme activity on DEAE-Sephadex chromatography differ with the three nuclear peaks, being found in ratios (uncorrected for the effect of increasing salt concentration) of 8:85:7 and the mitochondrial peaks in ratios of 8:32:60 at late log phase of growth under optimized conditions in which protease inhibitors and an antioxidant were included. The type of mitochondrial enzymes in 3-day-old cells differed from those grown to late logarithmic phase. It has been established that the enzymes of the mitochondrial preparation are associated with the membrane fraction. While extraction with 0.5 m KCl solubilizes considerable enzyme activity, greatly enhanced yields of enzyme MIII are obtained by addition of the antioxidant 2,6-di-t-butyl-4-hydroxymethyl phenol during enzyme extraction. Inhibition of protease activity has also been shown to have a major effect on the yield and distribution of enzymes obtained from mitochondrial preparations. The mitochondrial preparations of yeast polymerases are generally similar but not identical to corresponding nuclear polymerases in subunit molecular weights, inhibitor sensitivities, and in DNA template dependence. Comparative studies of nuclear and mitochondrial polymerases clearly establish that differences do exist among the isolated enzymes of these classes. It has not been ruled out to date that these enzymes may be derived in part or in total from the same cytoplasmic subunit pool, nor has it been established that any of these enzymes function in mitochondria in vivo.     

Partial reconstitution of human RNase P in HeLa cells between its RNA subunit with an affinity tag and the intact protein components

An RNA affinity tag was incorporated into the RNA subunit of human nuclear RNase P. The tagged RNA assembled with the protein components of RNase P inside HeLa cells to generate an active enzyme. Because of the specificity of the RNA tag to streptavidin, the reconstituted complex could be separated from the native enzyme and other ribonucleoproteins (particularly RNase MRP) by streptavidin agarose chromatography and could be recovered by the eluting agent, biotin. A mutant, tagged RNase P RNA, whose P3 domain was partially replaced, could not reconstitute with the proteins to yield an active enzyme. The P3 domain, therefore, is critical for the structure and function of RNase P.     

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

Ribonuclease P (RNase P) is a ribonucleoprotein that catalyzes the 5′ maturation of precursor transfer RNA in the presence of magnesium ions. The bacterial RNase P holoenzyme consists of one catalytically active RNA component and a single essential but catalytically inactive protein. In contrast, yeast nuclear RNase P is more complex with one RNA subunit and nine protein subunits. We have devised an affinity purification protocol to gently and rapidly purify intact yeast nuclear RNase P holoenzyme for transient kinetic studies. In pre-steady-state kinetic studies under saturating substrate concentrations, we observed an initial burst of tRNA formation followed by a slower, linear, steady-state turnover, with the burst amplitude equal to the concentration of the holoenzyme used in the reaction. These data indicate that the rate-limiting step in turnover occurs after pre-tRNA cleavage, such as mature tRNA release. Additionally, the steady-state rate constants demonstrate a large dependence on temperature that results in nonlinear Arrhenius plots, suggesting that a kinetically important conformational change occurs during catalysis. Finally, deletion of the 3′ trailer in pre-tRNA has little or no effect on the steady-state kinetic rate constants. These data suggest that, despite marked differences in subunit composition, the minimal kinetic mechanism for cleavage of pre-tRNA catalyzed by yeast nuclear RNase P holoenzyme is similar to that of the bacterial RNase P holoenzyme.     

Development of a single-nucleotide polymorphism (SNP) assay for genotyping of Pandora neoaphidis

Pandora neoaphidis (Entomophthoromycotina, Entomophthorales) is one of the most important fungal pathogens of aphids with great potential as a biological control agent. Development of tools that allow high-resolution monitoring of P. neoaphidis in the environment is a prerequisite for the successful implementation of biological control strategies. In this study, a single-nucleotide polymorphism (SNP) assay was developed. The assay targets 13 SNPs identified in 6 genomic regions including the largest subunit of nuclear RNA polymerase II (RPB1) gene, the second-largest subunit of nuclear RNA polymerase II (RPB2) gene, the β-tubulin (BTUB) gene, the elongation factor 1α-like (EFL) gene, the large subunit (LSU) rRNA gene, and the small subunit (SSU) rRNA gene together with the internal transcribed spacer (ITS). The assay allowed the discrimination of 15 different SNP profiles among 19 P. neoaphidis isolates and 4 P. neoaphidis-infected cadavers. Results showed that the assay is applicable to DNA extracted from infected aphids allowing genotyping of the fungus without cultivation. The SNP assay provides an efficient tool for investigation of population structures and dynamics of P. neoaphidis, as well as its persistence and epidemiology in agro-ecosystems. Furthermore, it constitutes a powerful approach for monitoring potential biological control strains of P. neoaphidis in the environment.     

Assembly of the Trypanosoma brucei 60S ribosomal subunit nuclear export complex requires trypanosome-specific proteins P34 and P37

We previously identified two Trypanosoma brucei RNA binding proteins, P34 and P37, and determined that they are essential for proper ribosomal assembly in this organism. Loss of these proteins via RNA interference is lethal and causes a decrease in both 5S rRNA levels and formation of 80S ribosomes, concomitant with a decrease in total cellular protein synthesis. These data suggest that these proteins are involved at some point in the ribosomal biogenesis pathway. In the current study, we have performed subcellular fractionation in conjunction with immune capture experiments specific for 60S ribosomal proteins and accessory factors in order to determine when and where P34 and P37 are involved in the ribosomal biogenesis pathway. These studies demonstrate that P34 and P37 associate with the 60S ribosomal subunit at the stage of the nucleolar 90S particle and remain associated subsequent to nuclear export. In addition, P34 and P37 associate with conserved 60S ribosomal subunit nuclear export factors exportin 1 and Nmd3, suggesting that they are components of the 60S ribosomal subunit nuclear export complex in T. brucei. Most significantly, the pre-60S complex does not associate with exportin 1 or Nmd3 in the absence of P34 and P37. These results demonstrate that, although T. brucei 60S ribosomal subunits utilize a nuclear export complex similar to that described for other organisms, trypanosome-specific factors are essential to the process.     

RNase MRP RNA and RNase P activity in plants are associated with a Pop1p containing complex

RNase P processes the 5'-end of tRNAs. An essential catalytic RNA has been demonstrated in Bacteria, Archaea and the nuclei of most eukaryotes; an organism-specific number of proteins complement the holoenzyme. Nuclear RNase P from yeast and humans is well understood and contains an RNA, similar to the sister enzyme RNase MRP. In contrast, no protein subunits have yet been identified in the plant enzymes, and the presence of a nucleic acid in RNase P is still enigmatic. We have thus set out to identify and characterize the subunits of these enzymes in two plant model systems. Expression of the two known Arabidopsis MRP RNA genes in vivo was verified. The first wheat MRP RNA sequences are presented, leading to improved structure models for plant MRP RNAs. A novel mRNA encoding the central RNase P/MRP protein Pop1p was identified in Arabidopsis, suggesting the expression of distinct protein variants from this gene in vivo. Pop1p-specific antibodies precipitate RNase P activity and MRP RNAs from wheat extracts. Our results provide evidence that in plants, Pop1p is associated with MRP RNAs and with the catalytic subunit of RNase P, either separately or in a single large complex.