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.     

Eukaryotic ribonuclease P: increased complexity to cope with the nuclear pre-tRNA pathway

Ribonuclease P is an ancient enzyme that cleaves pre-tRNAs to generate mature 5' ends. It contains an essential RNA subunit in Bacteria, Archaea, and Eukarya, but the degree to which the RNA subunit relies on proteins to supplement catalysis is highly variable. The eukaryotic nuclear holoenzyme has recently been found to contain almost twenty times the protein content of the bacterial enzymes, in addition to having split into at least two related enzymes with distinct substrate specificity. In this review, recent progress in understanding the molecular architecture and functions of nuclear forms of RNase P will be considered.     

Roles of protein subunits in RNA-protein complexes: lessons from ribonuclease P

Ribonucleoproteins (RNP) are involved in many essential processes in life. However, the roles of RNA and protein subunits in an RNP complex are often hard to dissect. In many RNP complexes, including the ribosome and the Group II introns, one main function of the protein subunits is to facilitate RNA folding. However, in other systems, the protein subunits may perform additional functions, and can affect the biological activities of the RNP complexes. In this review, we use ribonuclease P (RNase P) as an example to illustrate how the protein subunit of this RNP affects different aspects of catalysis. RNase P plays an essential role in the processing of the precursor to transfer RNA (pre-tRNA) and is found in all three domains of life. While every cell has an RNase P (ribonuclease P) enzyme, only the bacterial and some of the archaeal RNase P RNAs (RNA component of RNase P) are active in vitro in the absence of the RNase P protein. RNase P is a remarkable enzyme in the fact that it has a conserved catalytic core composed of RNA around which a diverse array of protein(s) interact to create the RNase P holoenzyme. This combination of highly conserved RNA and altered protein components is a puzzle that allows the dissection of the functional roles of protein subunits in these RNP complexes.     

RNase P: interface of the RNA and protein worlds

Ribonuclease P (RNase P) is an endonuclease involved in processing tRNA. It contains both RNA and protein subunits and occurs in all three domains of life: namely, Archaea, Bacteria and Eukarya. The RNase P RNA subunits from bacteria and some archaea are catalytically active in vitro, whereas those from eukaryotes and most archaea require protein subunits for activity. RNase P has been characterized biochemically and genetically in several systems, and detailed structural information is emerging for both RNA and protein subunits from phylogenetically diverse organisms. In vitro reconstitution of activity is providing insight into the role of proteins in the RNase P holoenzyme. Together, these findings are beginning to impart an understanding of the coevolution of the RNA and protein worlds.     

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.     

An essential protein-binding domain of nuclear RNase P RNA

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.     

RNA-dependent folding and stabilization of C5 protein during assembly of the E. coli RNase P holoenzyme

The pre-tRNA processing enzyme ribonuclease P is a ribonucleoprotein. In Escherichia coli assembly of the holoenzyme involves binding of the small (119 amino acid residue) C5 protein to the much larger (377 nucleotide) P RNA subunit. The RNA subunit makes the majority of contacts to the pre-tRNA substrate and contains the active site; however, binding of C5 stabilizes P RNA folding and contributes to high affinity substrate binding. Here, we show that RNase P ribonucleoprotein assembly also influences the folding of C5 protein. Thermal melting studies demonstrate that the free protein population is a mixture of folded and unfolded conformations under conditions where it assembles quantitatively with the RNA subunit. Changes in the intrinsic fluorescence of a unique tryptophan residue located in the folded core of C5 provide further evidence for an RNA-dependent conformational change during RNase P assembly. Comparisons of the CD spectra of the free RNA and protein subunits with that of the holoenzyme provide evidence for changes in P RNA structure in the presence of C5 as indicated by previous studies. Importantly, monitoring the temperature dependence of the CD signal in regions of the holoenzyme spectra that are dominated by protein or RNA structure permitted analysis of the thermal melting of the individual subunits within the ribonucleoprotein. These analyses reveal a significantly higher Tm for C5 when bound to P RNA and show that unfolding of the protein and RNA are coupled. These data provide evidence for a general mechanism in which the favorable free energy for formation of the RNA-protein complex offsets the unfavorable free energy of structural rearrangements in the RNA and protein subunits.     

Characterization of conserved sequence elements in eukaryotic RNase P RNA reveals roles in holoenzyme assembly and tRNA processing

RNase P is a ubiquitous endoribonuclease responsible for cleavage of the 5' leader of precursor tRNAs (pre-tRNAs). Although the protein composition of RNase P holoenzymes varies significantly among Bacteria, Archaea, and Eukarya, the holoenzymes have essential RNA subunits with several sequences and structural features that are common to all three kingdoms of life. Additional structural elements of the RNA subunits have been found that are conserved in eukaryotes, but not in bacteria, and might have functions specifically required by the more complex eukaryotic holoenzymes. In this study, we have mutated four eukaryotic-specific conserved regions in Saccharomyces cerevisiae nuclear RNase P RNA and characterized the effects of the mutations on cell growth, enzyme function, and biogenesis of RNase P. RNase P with mutations in each of the four regions tested is sufficiently functional to support life although growth of the resulting yeast strains was compromised to varying extents. Further analysis revealed that mutations in three different regions cause differential defects in holoenzyme assembly, localization, and pre-tRNA processing in vivo and in vitro. These data suggest that most, but not all, eukaryotic-specific conserved regions of RNase P RNA are important for the maturation and function of the holoenzyme.     

Interaction of Escherichia coli RNA polymerase with eukaryotic TATA-binding protein

Interaction with eukaryotic TATA-binding protein (TBP) was analyzed for natural Escherichia coli RNA polymerase or the recombinant holoenzyme, minimal enzyme, or its sigma subunit. Upon preincubation of full-sized RNA polymerase with TBP and further incubation with a constant amount of 32P-labeled phosphamide derivative of a TATA-containing oligodeoxyribonucleotide, the yield of the holoenzyme-oligonucleotide covalent complex decreased with increasing TBP concentration. This was considered as indirect evidence for complexing of RNA polymerase with TBP. In gel retardation assays, the holoenzyme, but neither minimal enzyme nor the sigma subunit, interacted with TPB, since the labeled probe formed complexes with both proteins in the reaction mixture combining TBP with the minimal enzyme or the sigma subunit. It was assumed that E. coli RNA polymerase is functionally similar to eukaryotic RNA polymerase II, and that the complete ensemble of all subunits is essential for the specific function of the holoenzyme.     

Interactions between subunits of Saccharomyces cerevisiae RNase MRP support a conserved eukaryotic RNase P/MRP architecture

Ribonuclease MRP is an endonuclease, related to RNase P, which functions in eukaryotic pre-rRNA processing. In Saccharomyces cerevisiae, RNase MRP comprises an RNA subunit and ten proteins. To improve our understanding of subunit roles and enzyme architecture, we have examined protein-protein and protein–RNA interactions in vitro, complementing existing yeast two-hybrid data. In total, 31 direct protein–protein interactions were identified, each protein interacting with at least three others. Furthermore, seven proteins self-interact, four strongly, pointing to subunit multiplicity in the holoenzyme. Six protein subunits interact directly with MRP RNA and four with pre-rRNA. A comparative analysis with existing data for the yeast and human RNase P/MRP systems enables confident identification of Pop1p, Pop4p and Rpp1p as subunits that lie at the enzyme core, with probable addition of Pop5p and Pop3p. Rmp1p is confirmed as an integral subunit, presumably associating preferentially with RNase MRP, rather than RNase P, via interactions with Snm1p and MRP RNA. Snm1p and Rmp1p may act together to assist enzyme specificity, though roles in substrate binding are also indicated for Pop4p and Pop6p. The results provide further evidence of a conserved eukaryotic RNase P/MRP architecture and provide a strong basis for studies of enzyme assembly and subunit function.     

Of proteins and RNA: The RNase P/MRP family

Nuclear ribonuclease (RNase) P is a ubiquitous essential ribonucleoprotein complex, one of only two known RNA-based enzymes found in all three domains of life. The RNA component is the catalytic moiety of RNases P across all phylogenetic domains; it contains a well-conserved core, whereas peripheral structural elements are diverse. RNA components of eukaryotic RNases P tend to be less complex than their bacterial counterparts, a simplification that is accompanied by a dramatic reduction of their catalytic ability in the absence of protein. The size and complexity of the protein moieties increase dramatically from bacterial to archaeal to eukaryotic enzymes, apparently reflecting the delegation of some structural functions from RNA to proteins and, perhaps, in response to the increased complexity of the cellular environment in the more evolutionarily advanced organisms; the reasons for the increased dependence on proteins are not clear. We review current information on RNase P and the closely related universal eukaryotic enzyme RNase MRP, focusing on their functions and structural organization.     

Molecular modeling of the three-dimensional structure of the bacterial RNase P holoenzyme

Bacterial ribonuclease P (RNase P), an enzyme involved in tRNA maturation, consists of a catalytic RNA subunit and a protein cofactor. Comparative phylogenetic analysis and molecular modeling have been employed to derive secondary and tertiary structure models of the RNA subunits from Escherichia coli (type A) and Bacillus subtilis (type B) RNase P. The tertiary structure of the protein subunit of B.subtilis and Staphylococcus aureus RNase P has recently been determined. However, an understanding of the structure of the RNase P holoenzyme (i.e. the ribonucleoprotein complex) is lacking. We have now used an EDTA-Fe-based footprinting approach to generate information about RNA-protein contact sites in E.coli RNase P. The footprinting data, together with results from other biochemical and biophysical studies, have furnished distance constraints, which in turn have enabled us to build three-dimensional models of both type A and B versions of the bacterial RNase P holoenzyme in the absence and presence of its precursor tRNA substrate. These models are consistent with results from previous studies and provide both structural and mechanistic insights into the functioning of this unique catalytic RNP complex.     

Footprinting analysis of interactions between the largest eukaryotic RNase P/MRP protein Pop1 and RNase P/MRP RNA components

Ribonuclease (RNase) P and RNase MRP are closely related catalytic ribonucleoproteins involved in the metabolism of a wide range of RNA molecules, including tRNA, rRNA, and some mRNAs. The catalytic RNA component of eukaryotic RNase P retains the core elements of the bacterial RNase P ribozyme; however, the peripheral RNA elements responsible for the stabilization of the global architecture are largely absent in the eukaryotic enzyme. At the same time, the protein makeup of eukaryotic RNase P is considerably more complex than that of the bacterial RNase P. RNase MRP, an essential and ubiquitous eukaryotic enzyme, has a structural organization resembling that of eukaryotic RNase P, and the two enzymes share most of their protein components. Here, we present the results of the analysis of interactions between the largest protein component of yeast RNases P/MRP, Pop1, and the RNA moieties of the enzymes, discuss structural implications of the results, and suggest that Pop1 plays the role of a scaffold for the stabilization of the global architecture of eukaryotic RNase P RNA, substituting for the network of RNA–RNA tertiary interactions that maintain the global RNA structure in bacterial RNase P.     

Protein-RNA interactions in the RNase P holoenzyme from Escherichia coli

The genes for the protein (C5 protein) and RNA (M1 RNA) subunits of Escherichia coli RNase P have been subcloned and their products prepared in milligram quantities by rapid procedures. The interactions between the two subunits of the enzyme have been studied in vitro by a filter-binding technique. The stoichiometry of the subunits in the holoenzyme is 1:1. The dissociation constant for the specific interactions of the subunits in the holoenzyme complex is approximately 4 x 10(-10) M. C5 protein also interacts with various RNA molecules in a non-specific manner with a dissociation constant of 2 x 10(-8) to 6 x 10(-8) M. Regions of M1 RNA required for interaction with C5 protein have been defined by deletion analysis and footprinting techniques. These interactions are localized primarily between nucleotides 82 to 96 and 170 to 270 of M1 RNA.     

The RNase P ofDictostyelium discoideum

Ribonuclease P (RNase P) is a key enzyme involved in tRNA biosynthesis. It catalyses the endonucleolytic cleavage of nearly all tRNA precursors to produce 5-end matured tRNA. RNase P activity has been found in all organisms examined, from bacteria to mammals. Eubacterial RNase P RNA is the only known RNA enzyme which functionsin trans in nature. Similar behaviour has not been demonstrated in RNase P enzymes examined from archaebacteria or eukaryotes. Characterisation of RNase P enzymes from more diverse eukaryotic species, including the slime moldDictyostelium discoideum, is useful for comparative analysis of the structure and function of eukaryotic RNase P.Abbreviations RNase P ribonuclease P - MN micrococcal nuclease     

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.     

Crystal structure of a C-terminal deletion mutant of human protein kinase CK2 catalytic subunit

Protein kinase CK2 (formerly called: casein kinase 2) is a heterotetrameric enzyme composed of two separate catalytic chains (CK2alpha) and a stable dimer of two non-catalytic subunits (CK2beta). CK2alpha is a highly conserved member of the superfamily of eukaryotic protein kinases. The crystal structure of a C-terminal deletion mutant of human CK2alpha was solved and refined to 2.5A resolution. In the crystal the CK2alpha mutant exists as a monomer in agreement with the organization of the subunits in the CK2 holoenzyme. The refined structure shows the helix alphaC and the activation segment, two main regions of conformational plasticity and regulatory importance in eukaryotic protein kinases, in active conformations stabilized by extensive contacts to the N-terminal segment. This arrangement is in accordance with the constitutive activity of the enzyme. By structural superimposition of human CK2alpha in isolated form and embedded in the human CK2 holoenzyme the loop connecting the strands beta4 and beta5 and the ATP-binding loop were identified as elements of structural variability. This structural comparison suggests that the ATP-binding loop may be the key region by which the non-catalytic CK2beta dimer modulates the activity of CK2alpha. The beta4/beta5 loop was found in a closed conformation in contrast to the open conformation observed for the CK2alpha subunits of the CK2 holoenzyme. CK2alpha monomers with this closed beta4/beta5 loop conformation are unable to bind CK2beta dimers in the common way for sterical reasons, suggesting a mechanism to protect CK2alpha from integration into CK2 holoenzyme complexes. This observation is consistent with the growing evidence that CK2alpha monomers and CK2beta dimers can exist in vivo independently from the CK2 holoenzyme and may possess physiological roles of their own.