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.

Ribonuclease P (RNase P) is a ribonucleoprotein that requires magnesium ions to catalyze the 5′ maturation of transfer RNA. To identify interactions essential for catalysis, the properties of RNase P containing single sulfur substitutions for nonbridging phosphodiester oxygens in helix P4 of Bacillus subtilis RNase P were analyzed using transient kinetic experiments. Sulfur substitution at the nonbridging oxygens of the phosphodiester bond of nucleotide U51 only modestly affects catalysis. However, phosphorothioate substitutions at A49 and G50 decrease the cleavage rate constant enormously (300–4,000-fold for P RNA and 500–15,000-fold for RNase P holoenzyme) in magnesium without affecting the affinity of pre-tRNAAsp, highlighting the importance of this region for catalysis. Furthermore, addition of manganese enhances pre-tRNA cleavage catalyzed by B. subtilis RNase P RNA containing an SP phosphorothioate modification at A49, as observed for Escherichia coli P RNA [Christian et al., RNA, 2000, 6:511–519], suggesting that an essential metal ion may be coordinated at this site. In contrast, no manganese rescue is observed for the A49 Sp phosphorothioate modification in RNase P holoenzyme. These differential manganese rescue effects, along with affinity cleavage, suggest that the protein component may interact with a metal ion bound near A49 in helix P4 of P RNA.

Catalytic complexes of nuclear ribonuclease P (RNase P) ribonucleoproteins are composed of several protein subunits that appear to have specific roles in enzyme function in tRNA processing. This review describes recent progress made in the characterization of human RNase P, its relationship with the ribosomal RNA processing ribonucleoprotein RNase MRP, and the unexpected evolutionary conservation of its subunits. A new model for the biosynthesis of human RNase P is presented, in which this process is dynamic, transcription-dependent, and implicates functionally distinct nuclear compartments in tRNA biogenesis.

Eukaryotic RNase P and RNase MRP are endoribonucleases composed of RNA and protein subunits. The RNA subunits of each enzyme share substantial secondary structural features, and most of the protein subunits are shared between the two. One of the conserved RNA subdomains, designated P3, has previously been shown to be required for nucleolar localization. Phylogenetic sequence analysis suggests that the P3 domain interacts with one of the proteins common to RNase P and RNase MRP, a conclusion strengthened by an earlier observation that the essential domain can be interchanged between the two enzymes. To examine possible functions of the P3 domain, four conserved nucleotides in the P3 domain of Saccharomyces cerevisiae RNase P RNA (RPR1) were randomized to create a library of all possible sequence combinations at those positions. Selection of functional genes in vivo identified permissible variations, and viable clones that caused yeast to exhibit conditional growth phenotypes were tested for defects in RNase P RNA and tRNA biosynthesis. Under nonpermissive conditions, the mutants had reduced maturation of the RPR1 RNA precursor, an expected phenotype in cases where RNase P holoenzyme assembly is defective. This loss of RPR1 RNA maturation coincided, as expected, with a loss of pre-tRNA maturation characteristic of RNase P defects. To test whether mutations at the conserved positions inhibited interactions with a particular protein, specific binding of the individual protein subunits to the RNA subunit was tested in yeast using the three-hybrid system. Pop1p, the largest subunit shared by RNases P and MRP, bound specifically to RPR1 RNA and the isolated P3 domain, and this binding was eliminated by mutations at the conserved P3 residues. These results indicate that Pop1p interacts with the P3 domain common to RNases P and MRP, and that this interaction is critical in the maturation of RNase P holoenzyme.

Ribonuclease P (RNase P) catalyzes the 5′ maturation of precursor tRNA transcripts and, in bacteria, is composed of a catalytic RNA and a protein. We investigated the oligomerization state and the shape of the RNA alone and the holoenzyme of Bacillus subtilis RNase P in the absence of substrate by synchrotron small-angle X-ray scattering and affinity retention. The B. subtilis RNase P RNA alone is a monomer; however, the scattering profile changes upon the addition of monovalent ions, possibly suggesting different interdomain angles. To our surprise, the X-ray scattering data combined with the affinity retention results indicate that the holoenzyme contains two RNase P RNA and two RNase P protein molecules. We propose a structural model of the holoenzyme with a symmetrical arrangement of the two RNA subunits, consistent with the X-ray scattering results. This (P RNA)2(P protein)2 complex likely binds substrate differently than the conventional (P RNA)1(P protein)1 complex; therefore, the function of the B. subtilis RNase P holoenzyme may be more diverse than previously thought. These revisions to our knowledge of the RNase P holoenzyme suggest a more versatile role for proteins in ribonucleoprotein complexes.

We investigated the catalytic efficiency and the specificity of the Bacillus subtilis RNase P holoenzyme reaction with substrates that contain a single strand, a hairpin loop, or a tRNA 3′ to the cleavage site. At a saturating ribozyme concentration, RNase P can cleave a single-stranded RNA at ∼0.6 min−1 at pH 7.8. Replacing the single-stranded RNA 3′ to the cleavage site by a hairpin loop or by the yeast tRNAPhe increases the cleavage rate by up to ∼600-fold and ∼3,200-fold, respectively. These results show that compared to a single-stranded RNA substrate, the cleavage rate for the holoenzyme reaction is primarily enhanced by an acceptor-stem-like helix. Substrate binding, ∼7–10 μM for a single-stranded RNA, improves by ∼1,000-fold upon the addition of the tRNA. The efficiency of the RNase P holoenzyme cleaving a single-stranded RNA is sufficiently high to consider autolytic processing of the RNase P RNA (denoted P RNA) transcript in the cell. The addition of the RNase P protein to a precursor form of the P RNA in vitro results in autolytic processing of the 5′ and the 3′ end of this precursor in a matter of minutes. Autolytic processing produces the reported 5′ end of the mature P RNA. The precise 3′ end generated by autolytic processing is different over the course of the reaction and the final product is 4 nt shorter than the reported 3′ end of the B. subtilis P RNA. The observed 3′ end in vitro is consistent with the property of the holoenzyme reaction with single-stranded RNA substrates. The discrepancy with the reported 3′ end may be due to other processing events in vivo or inaccurate determination of the mature 3′ end of the P RNA isolated from the cell. We propose that the mature B. subtilis P RNA is generated at least in part by autolytic processing upon the binding of the RNase P protein to the precursor P RNA.

We determined the solution structure of two 27-nt RNA hairpins and their complexes with cobalt(III)-hexammine (Co(NH3)63+) by NMR spectroscopy. The RNA hairpins used in this study are the P4 region from Escherichia coli RNase P RNA and a C-to-U mutant that confers altered divalent metal-ion specificity (Ca2+ replaces Mg2+) for catalytic activity of this ribozyme. Co(NH3)63+ is a useful spectroscopic probe for Mg(H2O)62+-binding sites because both complexes have octahedral symmetry and have similar radii. The thermodynamics of binding to both RNA hairpins was studied using chemical shift changes upon titration with Mg2+, Ca2+, and Co(NH3)63+. We found that the equilibrium binding constants for each of the metal ions was essentially unchanged when the P4 model RNA hairpin was mutated, although the NMR structures show that the RNA hairpins adopt different conformations. In the C-to-U mutant a C[bull ]G base pair is replaced by U[bull ]G, and the conserved bulged uridine in the P4 wild-type stem shifts in the 3′ direction by 1 nt. Intermolecular NOE cross-peaks between Co(NH3)63+ and RNA protons were used to locate the site of Co(NH3)63+ binding to both RNA hairpins. The metal ion binds in the major groove near a bulge loop, but is shifted 5′ by more than 1 bp in the mutant. The change of the metal-ion binding site provides a possible explanation for changes in catalytic activity of the mutant RNase P in the presence of Ca2+.

RNase P ribozyme cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural ptRNA substrate. When covalently linked with a guide sequence, the ribozyme can function as a sequence-specific endonuclease and cleave any target RNA sequences that base pair with the guide sequence. Using a site-directed ultraviolet (UV) cross-linking approach, we have mapped the regions of the ribozyme that are in close proximity to a substrate that contains the mRNA sequence encoding thymidine kinase of human herpes simplex virus 1. Our data suggest that the cleavage site of the mRNA substrate is positioned at the same regions of the ribozyme that bind to the cleavage site of a ptRNA. The mRNA-binding domains include regions that interact with the acceptor stem and T-stem and in addition, regions that are unique and not in close contact with a ptRNA. Identification of the mRNA-binding site provides a foundation to study how RNase P ribozymes achieve their sequence specificity and facilitates the development of gene-targeting ribozymes.

The eukaryotic nucleolus contains a large number of small RNA molecules that, in the form of small nucleolar ribonucleoprotein complexes (snoRNPs), are involved in the processing and modification of pre-rRNA. One of the snoRNPs that has been shown to possess enzymatic activity is the RNase MRP. RNase MRP is an endoribonuclease involved in the formation of the 5′ end of 5.8S rRNA. In this study the association of the hPop1 protein with the RNase MRP complex was investigated. The hPop1 protein seems not to be directly bound to the RNA component, but requires nt 1–86 and 116–176 of the MRP RNA to associate with the RNase MRP complex via protein–protein interactions. UV crosslinking followed by ribonuclease treatment and immunoprecipitation with anti-Th/To antibodies revealed three human proteins of about 20, 25, and 40 kDa that can associate with the RNase MRP complex. The 20- and 25-kDa proteins appear to bind to stem-loop I of the MRP RNA whereas the 40-kDa protein requires the central part of the MRP RNA (nt 86–176) for association with the RNase MRP complex. In addition, we show that the human RNase P proteins Rpp30 and Rpp38 are also associated with the RNase MRP complex. Expression of Vesicular Stomatitis Virus- (VSV) tagged versions of these proteins in HeLa cells followed by anti-VSV immunoprecipitation resulted in coprecipitation of both RNase P and RNase MRP complexes. Furthermore, UV crosslinking followed by anti-Th/To and anti-Rpp38 immunoprecipitation revealed that the 40-kDa protein we detected in UV crosslinking is probably identical to Rpp38.

External guide sequences (EGSs) are small RNA molecules which consist of a sequence complementary to a target mRNA and render the target RNA susceptible to degradation by ribonuclease P (RNase P). EGSs were designed to target the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1 for degradation. These EGSs were shown to be able to direct human RNase P to cleave the TK mRNA sequence efficiently in vitro. A reduction of about 80% in the expression level of both TK mRNA and protein was observed in human cells that steadily expressed an EGS, but not in cells that either did not express the EGS or produced a “disabled” EGS which carried a single nucleotide mutation that precluded RNase P recognition. Thus, EGSs may represent novel gene-targeting agents for inhibition of gene expression and antiviral activity.

We have studied the structure and divalent metal ion binding of a domain of the ribozyme RNase P RNA that is involved in base pairing with its substrate. Our data suggest that the folding of this internal loop, the P15-loop, is similar irrespective of whether it is part of the full-length ribozyme or part of a model RNA molecule. We also conclude that this element constitutes an autonomous divalent metal ion binding domain of RNase P RNA and our data suggest that certain specific chemical groups within the P15-loop participate in coordination of divalent metal ions. Substitutions of the Sp- and Rp-oxygens with sulfur at a specific position in this loop result in a 2.5–5-fold less active ribozyme, suggesting that Mg2+ binding at this position contributes to function. Our findings strengthen the concept that small RNA building blocks remain basically unchanged when removed from their structural context and thus can be used as models for studies of their potential function and structure within native RNA molecules.