Recent insights into the structure and function of the ribonucleoprotein enzyme ribonuclease P

In bacteria, the tRNA-processing endonuclease ribonuclease P is composed of a large ( approximately 400 nucleotide) catalytic RNA and a smaller ( approximately 100 amino acid) protein subunit that is essential for substrate recognition. Current biochemical and biophysical investigations are providing fresh insights into the modular architecture of the ribozyme, the mechanisms of substrate specificity and the role of essential metal ions in catalysis. Together with recent high-resolution structures of portions of the ribozyme, these findings are beginning to reveal how the functions of RNA and protein are coordinated in this ribonucleoprotein enzyme.     

Ribonuclease P: a ribonucleoprotein enzyme

The ribonucleoprotein ribonuclease P catalyzes the hydrolysis of a specific phosphodiester bond in precursor tRNA to form the mature 5' end of tRNA. Recent studies have shed light on the structures of RNase-P-RNA-P-protein and RNase-P-RNA-precursor-tRNA complexes, as well as on the positions of catalytic metal ions, emphasizing the importance of the structure to the catalytic function.     

A small RNA that complements mutants in the RNA processing enzyme ribonuclease P

Four temperature-sensitive RNase P mutants were analyzed for the accumulation of 10 S RNA. In the 10 S region of the polyacrylamide gel two molecules appear, a and b. While the level of 10 Sa seems to be affected in some of the mutants, the 10 Sb molecule was not found in rnpB mutants. A plasmid (pL2), which contains Escherichia coli DNA sequences that complement, at least partially, rnp mutations, directs the synthesis of 10 Sb RNA. The presence of the pL2 plasmid complements the rnpA49, rnpB3187 and the rnpC241 mutations, as revealed by colony formation at “non-permissive” temperatures. However, the complementation of the rnpA49 mutation is much better than that of the other mutations. The complementation can also be measured by the increased level of RNase P activity in extracts. 10 Sa and b RNAs are unique among all RNAs tested thus far, since they are stable during exponential growth at 30 °C and 37 °C. However, at higher temperatures, such as 43 °C, the molecules are somewhat less stable, and they become rather labile when RNA synthesis is blocked by rifampicin. Structural analysis revealed that the 10 Sa and 10 Sb RNA molecules have dissimilar sequences.     

Comparative structure analysis of vertebrate ribonuclease P RNA.

Ribonuclease P cleaves 5'-precursor sequences from pre-tRNAs. All cellular RNase P holoenzymes contain homologous RNA elements; the eucaryal RNase P RNA, in contrast to the bacterial RNA, is catalytically inactive in the absence of the protein component(s). To understand the function of eucaryal RNase P RNA, knowledge of its structure is needed. Considerable effort has been devoted to comparative studies of the structure of this RNA from diverse organisms, including eucaryotes, primarily fungi, but also a limited set of vertebrates. The substantial differences in the sequences and structures of the vertebrate RNAs from those of other organisms have made it difficult to align the vertebrate sequences, thus limiting comparative studies. To expand our understanding of the structure of diverse RNase P RNAs, we have isolated by PCR and sequenced 13 partial RNase P RNA genes from 11 additional vertebrate taxa representing most extant major vertebrate lineages. Based on a recently proposed structure of the core elements of RNase P RNA, we aligned the sequences and propose a minimum consensus secondary structure for the vertebrate RNase P RNA.     

Conservation of helical structure contributes to functional metal ion interactions in the catalytic domain of ribonuclease P RNA

Like protein enzymes, catalytic RNAs contain conserved structure motifs important for function. A universal feature of the catalytic domain of ribonuclease P RNA is a bulged-helix motif within the P1-P4 helix junction. Here, we show that changes in bulged nucleotide identity and position within helix P4 affect both catalysis and substrate binding, while a subset of the mutations resulted only in catalytic defects. We find that the proximity of the bulge to sites of metal ion coordination in P4 is important for catalysis; moving the bulge distal to these sites and deleting it had similarly large effects, while moving it proximal to these sites had only a moderate effect on catalysis. To test whether the effects of the mutations are linked to metal ion interactions, we used terbium-dependent cleavage of the phosphate backbone to probe metal ion-binding sites in the wild-type and mutant ribozymes. We detect cleavages at specific sites within the catalytic domain, including helix P4 and J3/4, which have previously been shown to participate directly in metal ion interactions. Mutations introduced into P4 cause local changes in the terbium cleavage pattern due to alternate metal ion-binding configurations with the helix. In addition, a bulge deletion mutation results in a 100-fold decrease in the single turnover cleavage rate constant at saturating magnesium levels, and a reduced affinity for magnesium ions important for catalysis. In light of the alternate terbium cleavage pattern in P4 caused by bulge deletion, this decreased ability to utilize magnesium ions for catalysis appears to be due to localized structural changes in the ribozyme's catalytic core that weaken metal ion interactions in P4 and J3/4. The information reported here, therefore, provides evidence that the universal conservation of the P4 structure is based in part on optimization of metal ion interactions important for catalysis.     

Evidence for a polynuclear metal ion binding site in the catalytic domain of ribonuclease P RNA

Interactions with divalent metal ions are essential for the folding and function of the catalytic RNA component of the tRNA processing enzyme ribonuclease P (RNase P RNA). However, the number and location of specific metal ion interactions in this large, highly structured RNA are poorly understood. Using atomic mutagenesis and quantitative analysis of thiophilic metal ion rescue we provide evidence for metal ion interactions at the pro-R(P) and pro-S(P) non-bridging phosphate oxygens at nucleotide A67 in the universally conserved helix P4. Moreover, second-site modifications within helix P4 and the adjacent single stranded region (J3/4) provide the first evidence for metal ion interactions with nucleotide base functional groups in RNase P RNA and reveal the presence of an additional metal ion important for catalytic function. Together, these data are consistent with a cluster of metal ion interactions in the P1-P4 multi-helix junction that defines the catalytic core of the RNase P ribozyme.     

Mycoplasma fermentans simplifies our view of the catalytic core of ribonuclease P RNA.

The catalytic RNA moiety of (eu)bacterial RNase P is responsible for cleavage of the 5' leader sequence from precursor tRNAs. We report the sequence, the catalytic properties, and a phylogenetic-comparative structural analysis of the RNase P RNA from Mycoplasma fermentans, at 276 nt the smallest known RNase P RNA. This RNA is noteworthy in that it lacks a stem-loop structure (helix P12) that was thought previously to be universally present in bacterial RNase P RNAs. This finding suggests that helix P12 is not required for catalytic activity in vivo. In order to test this possibility in vitro, the kinetic properties of M. fermentans RNase P RNA and a mutant Escherichia coli RNase P RNA that was engineered to lack helix P12 were determined. These RNase P RNAs are catalytically active with efficiencies (Kcat/Km) comparable to that of native E. coli RNase P RNA. These results show that helix P12 is dispensable in vivo in some organisms, and therefore is unlikely to be essential for the mechanism of RNase P action. The notion that all phylogenetically volatile structures in RNase P RNA are dispensable for the catalytic mechanism was tested. A synthetic RNA representing the phylogenetic minimum RNase P RNA was constructed by deleting all evolutionarily variable structures from the M. fermentans RNA. This simplified RNA (Micro P RNA) was catalytically active in vitro with approximately 600-fold decrease in catalytic efficiency relative to the native RNA.     

Comparative analysis of ribonuclease P RNA structure in Archaea.

Although the structure of the catalytic RNA component of ribonuclease P has been well characterized in Bacteria, it has been little studied in other organisms, such as the Archaea. We have determined the sequences encoding RNase P RNA in eight euryarchaeal species: Halococcus morrhuae, Natronobacterium gregoryi, Halobacterium cutirubrum, Halobacteriurn trapanicum, Methanobacterium thermoautotrophicum strains deltaH and Marburg, Methanothermus fervidus and Thermococcus celer strain AL-1. On the basis of these and previously available sequences from Sulfolobus acidocaldarius, Haloferax volcanii and Methanosarcina barkeri the secondary structure of RNase P RNA in Archaea has been analyzed by phylogenetic comparative analysis. The archaeal RNAs are similar in both primary and secondary structure to bacterial RNase P RNAs, but unlike their bacterial counterparts these archaeal RNase P RNAs are not by themselves catalytically proficient in vitro.     

Phylogenetic comparative analysis and the secondary structure of ribonuclease P RNA--a review

The most incisive a priori approach to inferring the higher order structure of large RNAs has proven to be the use of phylogenetic comparisons. This article provides guidelines to the method, using as an illustration the elucidation of the secondary structure of the catalytic RNA subunit of ribonuclease P (RNase P). The resultant structure is compared to the possibilities that are predicted thermodynamically for the RNase P RNA sequences of nine eubacteria.     

Function and subnuclear distribution of Rpp21, a protein subunit of the human ribonucleoprotein ribonuclease P

Rpp21, a protein subunit of human nuclear ribonuclease P (RNase P) was cloned by virtue of its homology with Rpr2p, an essential subunit of Saccharomyces cerevisiae nuclear RNase P. Rpp21 is encoded by a gene that resides in the class I gene cluster of the major histocompatibility complex, is associated with highly purified RNase P, and binds precursor tRNA. Rpp21 is predominantly localized in the nucleoplasm but is also observed in nucleoli and Cajal bodies when expressed at high levels. Intron retention and splice-site selection in Rpp21 precursor mRNA regulate the intranuclear distribution of the protein products and their association with the RNase P holoenzyme. Our study reveals that dynamic nuclear structures that include nucleoli, the perinucleolar compartment and Cajal bodies are all involved in the production and assembly of human RNase P.     

Folding of a universal ribozyme: the ribonuclease P RNA

Ribonuclease P is among the first ribozymes discovered, and is the only ubiquitously occurring ribozyme besides the ribosome. The bacterial RNase P RNA is catalytically active without its protein subunit and has been studied for over two decades as a model system for RNA catalysis, structure and folding. This review focuses on the thermodynamic, kinetic and structural frameworks derived from the folding studies of bacterial RNase P RNA.     

The structure of ribonuclease P protein from Staphylococcus aureus reveals a unique binding site for single-stranded RNA

Ribonuclease P (RNaseP) catalyses the removal of the 5'-leader sequence from pre-tRNA to produce the mature 5' terminus. The prokaryotic RNaseP holoenzyme consists of a catalytic RNA component and a protein subunit (RNaseP protein), which plays an auxiliary but essential role in vivo by binding to the 5'-leader sequence and broadening the substrate specificity of the ribozyme.We determined the three-dimensional high-resolution structure of the RNaseP protein from Staphylococcus aureus (117 amino acid residues) by nuclear magnetic resonance (NMR) spectroscopy in solution. The protein has an alphabeta-fold, similar to the ribonucleoprotein domain. We used small nucleic acid molecules as a model for the 5'-leader sequence to probe the propensity for generic single-stranded RNA binding on the protein surface. The NMR results reveal a contiguous interaction site, which is identical with the previously identified leader sequence binding site in RNaseP holoenzyme. The conserved arginine-rich motif does not bind single-stranded RNA. It is likely that this peptide segment binds selectively to double-stranded sections of P RNA, which are conformationally more rigid. Given the essentiality of RNaseP for the viability of the organism, knowledge of the S. aureus protein structure and insight into its interaction with RNA will help us to develop RNaseP and RNaseP protein as targets for novel antibiotics against this pathogen.     

Eukaryotic RNase P: role of RNA and protein subunits of a primordial catalytic ribonucleoprotein in RNA-based catalysis

Ribonuclease P (RNase P) is an essential enzyme that processes the 5' leader sequence of precursor tRNA. Eubacterial RNase P is an RNA enzyme, while its eukaryotic counterpart acts as catalytic ribonucleoprotein, consisting of RNA and numerous protein subunits. To study the latter form, we reconstitute human RNase P activity, demonstrating that the subunits H1 RNA, Rpp21, and Rpp29 are sufficient for 5' cleavage of precursor tRNA. The reconstituted RNase P precisely delineates its cleavage sites in various substrates and hydrolyzes the phosphodiester bond. Rpp21 and Rpp29 facilitate catalysis by H1 RNA, which seems to require a phylogenetically conserved pseudoknot structure for function. Unexpectedly, Rpp29 forms a catalytic complex with M1 RNA of E. coli RNase P. The results uncover the core components of eukaryotic RNase P, reveal its evolutionary origin in translation, and provide a paradigm for studying RNA-based catalysis by other nuclear and nucleolar ribonucleoprotein enzymes.     

The iStem, a long-range RNA secondary structure element required for efficient exon inclusion in the Drosophila Dscam pre-mRNA

The Drosophila Dscam gene encodes 38,016 different proteins, due to alternative splicing of 95 of its 115 exons, that function in axon guidance and innate immunity. The alternative exons are organized into four clusters, and the exons within each cluster are spliced in a mutually exclusive manner. Here we describe an evolutionarily conserved RNA secondary structure we call the Inclusion Stem (iStem) that is required for efficient inclusion of all 12 variable exons in the exon 4 cluster. Although the iStem governs inclusion or exclusion of the entire exon 4 cluster, it does not play a significant role in determining which variable exon is selected. Thus, the iStem is a novel type of regulatory element that simultaneously controls the splicing of multiple alternative exons.     

The secondary structure and poly(A) content of globin messenger RNA as a pure RNA and in polyribosome-derived ribonucleoprotein complexes.

The conformation in solution of duck and rabbit globin mRNA, and of the duck mRNA in the mRNA - protein particle, has been investigated by optical methods and also by the use of the dye ethidium bromide which becomes highly fluorescent when intercalated into the double-stranded regions of a nucleic acid. On the basis of the properties of this dye and on the ability of homopolyribonucleotides to form double-stranded structures we have, in addition, developed a simple and sensitive assay for the detection and quantitisation of sequences rich in a particular residue that may be present in an RNA chain. In solution, 45 to 60% of the nucleotides of duck globin nRNA were found to be in bihelical regions. A similar degree of secondary structure was found in rabbit globin mRNA (this paper), as well as in calf lens mRNA and mRNAs from ewe mammary gland (other results). All samples of globin mRNA examined in this work containeda sequence of poly(A), which has poly(U) binding properties similar to that of synthetic poly(a): no specific interaction between the poly(A) sequence and the rest of the molecules can be detected. The fraction of adenosine residues within these poly(A) segments represents 4% in rabbit mRNA and 8 to 9% in duck mRNA. An additional adenosine-rich segment interspersed with guanosine and possibly other residues, was also detected in one duck mRNA sample. The RNA in the duck mRNA - protein particle is also highly structured. The melting profile in the range of 20 to 65 degrees C is quite similar to that of free mRNA and the ability of ethidium bromide to intercalate is reduced to the extent of 70%. Yet the dichroic spectra of free and bound mRNA are significantly distinct. These data suggest that free and protein-bound mRNA May have a very similar degree of secondary structure but with distinct detailed conformation in bihelical regions (change in base tilting for example). Direct evidence has been obtained that proteins stick to the poly(A) segment in the particle since the fraction of adenosine residues detectable by our poly(u) titration procedure is reduced to 50% of that observed in the free mRNA.     

Lead-catalyzed cleavage of ribonuclease P RNA as a probe for integrity of tertiary structure.

Pb(2+)-catalyzed cleavage of RNA has been shown previously to be a useful probe for tertiary structure. In the present study, Pb2+ cleavage patterns were identified for ribonuclease P RNAs from three phylogenetically disparate organisms, Escherichia coli, Chromatium vinosum, Bacillus subtilis, and for E. coli RNase P RNAs that had been altered by deletions. Each of the native RNAs undergoes cleavage at several sites in the core structure that is common to all bacterial RNase P RNAs. All the cleavages occur in non-paired regions of the secondary structure models of the RNAs, in regions likely to be involved in tertiary interactions. Two cleavage sites occur at homologous positions in all the native RNAs, regardless of sequence variation, suggesting common tertiary structural features. The Pb2+ cleavage sites in four deletion mutants of E. coli RNase P RNA differed from the native pattern, indicating alterations in the tertiary structures of the mutant RNAs. This conclusion is consistent with previously characterized properties of the mutant RNAs. The Pb2+ cleavage assay is thus a useful probe to reveal alteration of tertiary structure in RNase P RNA.     

Cloning the gene for ribonuclease E, an RNA processing enzyme

A transducing bacteriophage lambda Ch25rne+, which codes for ribonuclease E of E. coli, has been isolated. To achieve this a random library of Escherichia coli HindIII fragments was cloned in the lambda Charon 25 vector (prepared in F.R. Blattner's laboratory), and lambda Ch25rne+ was selected by its ability upon lysogenization to enable a temperature-sensitive (ts) rne-3071 mutant to grow and to exhibit normal RNA processing at the nonpermissive temperature of 45 degrees C. The level of RNase E was doubled in an rne+ strain lysogenized with lambda Ch25rne+. lambda Ch25rne+ directs the synthesis of a polypeptide of 71 000 m.wt., which is the size of RNase E. Restriction analysis and electron micrography of heteroduplexes suggested that the size of the host DNA insert is about 1.9 kb.     

Phylogenetic-comparative analysis of the eukaryal ribonuclease P RNA

Ribonuclease P (RNase P) is the ribonucleoprotein enzyme that cleaves 5'-leader sequences from precursor-tRNAs. Bacterial and eukaryal RNase P RNAs differ fundamentally in that the former, but not the latter, are capable of catalyzing pre-tRNA maturation in vitro in the absence of proteins. An explanation of these functional differences will be assisted by a detailed comparison of bacterial and eukaryal RNase P RNA structures. However, the structures of eukaryal RNase P RNAs remain poorly characterized, compared to their bacterial and archaeal homologs. Hence, we have taken a phylogenetic-comparative approach to refine the secondary structures of eukaryal RNase P RNAs. To this end, 20 new RNase P RNA sequences have been determined from species of ascomycetous fungi representative of the genera Arxiozyma, Clavispora, Kluyveromyces, Pichia, Saccharomyces, Saccharomycopsis, Torulaspora, Wickerhamia, and Zygosaccharomyces. Phylogenetic-comparative analysis of these and other sequences refines previous eukaryal RNase P RNA secondary structure models. Patterns of sequence conservation and length variation refine the minimum-consensus model of the core eukaryal RNA structure. In comparison to bacterial RNase P RNAs, the eukaryal homologs lack RNA structural elements thought to be critical for both substrate binding and catalysis. Nonetheless, the eukaryal RNA retains the main features of the catalytic core of the bacterial RNase P. This indicates that the eukaryal RNA remains intrinsically a ribozyme.