RNase MRP and RNase P share a common substrate.

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that processes RNA from the mammalian mitochondrial displacement loop containing region. RNase P is a site-specific ribonucleoprotein endoribonuclease that processes pre-tRNAs to generate their mature 5'-ends. A similar structure for the RNase P and RNase MRP RNAs and a common cleavage mechanism for RNase MRP and RNase P enzymes have been proposed. Experiments with protein synthesis antibiotics have shown that both RNase MRP and RNase P are inhibited by puromycin. We also show that E. coli RNase P cleaves the RNase MRP substrate, mouse mitochondrial primer RNA, exactly at a site that is cleaved by RNase MRP.     

The yeast,Saccharomyces cerevisiae,RNase P/MRP ribonucleoprotein endoribonuclease family

Ribonuclease P (RNase P) is a ribonucleoprotein responsible for the endonucleolytic cleavage of the 5-termini of tRNAs. Ribonuclease MRP (RNase MRP) is a ribonucleoprotein that has the ability to cleave both mitochondrial RNA primers presumed to be involved in mitochondrial DNA replication and rRNA precursors for the production of mature rRNAs. Several lines of evidence suggest that these two ribonucleoproteins are related to each other, both functionally and evolutionarily. Both of these enzymes have activity in the nucleus and mitochondria. Each cleave their RNA substrates in a divalent cation dependent manner to generate 5-phosphate and 3-OH termini. In addition, the RNA subunits of both complexes can be folded into a similar secondary structure. Each can be immunoprecipitated from mammalian cells with Th antibodies. In yeast, both have been found to share at least one common protein. This review will discuss some of the recent advances in our understanding of the structure, function and evolutionary relationship of these two enzymes in the yeast,Saccharomyces cerevisiae.Abbreviations LRI long range interaction - mt mitochondrial - MRP mitochondrial RNA processing - NME nuclear mitochondrial endonuclease - POP processing of precursor - RNase ribonuclease - SNM suppressor of NME - RNP ribonucleoprotein     

Structural organizations of yeast RNase P and RNase MRP holoenzymes as revealed by UV-crosslinking studies of RNA-protein interactions

Eukaryotic ribonuclease (RNase) P and RNase MRP are closely related ribonucleoprotein complexes involved in the metabolism of various RNA molecules including tRNA, rRNA, and some mRNAs. While evolutionarily related to bacterial RNase P, eukaryotic enzymes of the RNase P/MRP family are much more complex. Saccharomyces cerevisiae RNase P consists of a catalytic RNA component and nine essential proteins; yeast RNase MRP has an RNA component resembling that in RNase P and 10 essential proteins, most of which are shared with RNase P. The structural organizations of eukaryotic RNases P/MRP are not clear. Here we present the results of RNA-protein UV crosslinking studies performed on RNase P and RNase MRP holoenzymes isolated from yeast. The results indicate locations of specific protein-binding sites in the RNA components of RNase P and RNase MRP and shed light on the structural organizations of these large ribonucleoprotein complexes.     

Modular architecture of eukaryotic RNase P and RNase MRP revealed by electron microscopy

Ribonuclease P (RNase P) and RNase MRP are closely related ribonucleoprotein enzymes, which process RNA substrates including tRNA precursors for RNase P and 5.8 S rRNA precursors, as well as some mRNAs, for RNase MRP. The structures of RNase P and RNase MRP have not yet been solved, so it is unclear how the proteins contribute to the structure of the complexes and how substrate specificity is determined. Using electron microscopy and image processing we show that eukaryotic RNase P and RNase MRP have a modular architecture, where proteins stabilize the RNA fold and contribute to cavities, channels and chambers between the modules. Such features are located at strategic positions for substrate recognition by shape and coordination of the cleaved-off sequence. These are also the sites of greatest difference between RNase P and RNase MRP, highlighting the importance of the adaptation of this region to the different substrates.     

RNA-protein interactions in the human RNase MRP ribonucleoprotein complex

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.     

Interactions of a Pop5/Rpp1 heterodimer with the catalytic domain of RNase MRP

Ribonuclease (RNase) MRP is a multicomponent ribonucleoprotein complex closely related to RNase P. RNase MRP and eukaryotic RNase P share most of their protein components, as well as multiple features of their catalytic RNA moieties, but have distinct substrate specificities. While RNase P is practically universally found in all three domains of life, RNase MRP is essential in eukaryotes. The structural organizations of eukaryotic RNase P and RNase MRP are poorly understood. Here, we show that Pop5 and Rpp1, protein components found in both RNase P and RNase MRP, form a heterodimer that binds directly to the conserved area of the putative catalytic domain of RNase MRP RNA. The Pop5/Rpp1 binding site corresponds to the protein binding site in bacterial RNase P RNA. Structural and evolutionary roles of the Pop5/Rpp1 heterodimer in RNases P and MRP are discussed.     

Architecture and function of the human endonucleases RNase P and RNase MRP

In the past decade, important advances have been made in our knowledge of the composition of human RNase MRP and RNase P complexes. Both ribonucleoprotein particles function as endonucleases and contain RNA components that are structurally related. RNase MRP has been suggested to be involved in the processing of precursor rRNA; RNase P, in the maturation of tRNA. Here we give an overview of current data on the structure and function of human RNase MRP and RNase P particles, with emphasis on their molecular composition. At present, seven protein subunits, probably all associated with both ribonucleoprotein particles, have been isolated and their corresponding cDNAs cloned. Although no known structural motifs can be identified in the amino acid sequences of these proteins, the majority is clearly rich in basic residues. For two protein subunits, a cluster of basic amino acids have been shown to be involved in nucleolar accumulation, whereas another protein, which lacks such a region, probably enters the nucleolus by way of a piggyback mechanism. The binding regions for several of the protein subunits on the RNA have been identified, and the data have been used to create a putative structural model for the RNase MRP particle. The rather obscure situation concerning the association of the autoantigenic Th-40 protein and its possible relationship with one of the subunits, Rpp38, is discussed.     

Structural and functional similarities between MRP and RNase P

RNase P, the enzyme responsible for 5-end processing of tRNAs and 4.5S RNA, has been extensively characterized fromE. coli. The RNA component ofE. coli RNase P, without the protein, has the enzymatic activity and is the first true RNA enzyme to be characterized. RNase P and MRP are two distinct nuclear ribonucleoprotein (RNP) particles characterized in many eukaryotic cells including human, yeast and plant cells. There are many similarities between RNase P and MRP. These include: (1) sequence specific endonuclease activity; (2) homology at the primary and secondary structure levels; and (3) common proteins in both the RNPs. It is likely that RNase P and MRP originated from a common ancestor.     

Molecular modeling of the three-dimensional architecture of the RNA component of yeast RNase MRP.

RNase mitochondrial RNA processing (MRP) is a ribonucleoprotein endoribonuclease that is involved in RNA processing events in both the nucleus and the mitochondria. The MRP RNA is both structurally and evolutionarily related to RNase P, the ribonucleoprotein endoribonuclease that processes the 5'-end of tRNAs. Previous analysis of the RNase MRP RNA by phylogenetic analysis and chemical modification has revealed strikingly conserved secondary structural elements in all characterized RNase MRP RNAs. Utilizing successive constraint modeling and energy minimization I derived a three-dimensional model of the yeast RNase MRP RNA. The final model predicts several notable features. First, the enzyme appears to contain two separate structural domains, one that is highly conserved among all MRP and P RNAs and a second that is only conserved in MRP RNAs. Second, nearly all of the highly conserved nucleotides cluster in the first domain around a long-range interaction (LRI-I). This LRI-I is characterized by a ubiquitous uridine base, which points into a cleft between these two structural domains generating a potential active site for RNA cleavage. Third, helices III and IV (the yeast equivalent of the To-binding site) model as a long extended helix. This region is believed to be the binding site of shared proteins between RNase P and RNase MRP and would provide a necessary platform for binding these seven proteins. Indeed, several residues conserved between the yeast MRP and P RNAs cluster in the central region of these helixes. Lastly, characterized mutations in the MRP RNA localize in the model based on their severity. Those mutations with little or no effect on the activity of the enzyme localize to the periphery of the model, while the most severe mutations localize to the central portion of the molecule where they would be predicted to cause large structural defects. Press.     

hPop5, a protein subunit of the human RNase MRP and RNase P endoribonucleases.

The RNase MRP and RNase P particles both function as endoribonucleases. RNase MRP has been implicated in the processing of precursor-rRNA, whereas RNase P has been shown to function in the processing of pre-tRNA. Both ribonucleoprotein particles have an RNA component that can be folded into a similar secondary structure and share several protein components. We have identified human, rat, mouse, cow, and Drosophila homologues of the Pop5p protein subunit of the yeast RNase MRP and RNase P complexes. The human Pop5 cDNA encodes a protein of 163 amino acids with a predicted molecular mass of 18.8 kDa. Polyclonal antibodies raised against recombinant hPop5 identified a 19-kDa polypeptide in HeLa cells and showed that hPop5 is associated with both RNase MRP and RNase P. Using affinity-purified anti-hPop5 antibodies, we demonstrated that the endogenous hPop5 protein is localized in the nucleus and accumulates in the nucleolus, which is consistent with its association with RNase MRP and RNase P. Catalytically active RNase P was partially purified from HeLa cells, and hPop5 was shown to be associated with it. Finally, the evolutionarily conserved acidic C-terminal tail of hPop5 appeared to be required neither for complex formation nor for RNase P activity.     

Further characterization of human RNase MRP/RNase P and related autoantibodies

We characterized a panel of human RNase MRP/RNase P autoantibodies by immunoprecipitation, immunodepletion, immunoaffinity purification and immunoblotting. We report on the protein spectrum that is recognized by RNase MRP/RNase P autoantibodies. We also describe another, related patient serum that based on these assays does not immunoprecipitate RNase P/MRP/Th40. This autoantibody KC, however, coimmunoprecipitates the RNase MRP/RNase P associated RNAs from HeLa and La9 cell extracts as shown by nuclease protection experiments.     

hPop4: a new protein subunit of the human RNase MRP and RNase P ribonucleoprotein complexes.

RNase MRP is a ribonucleoprotein particle involved in the processing of pre-rRNA. The RNase MRP particle is structurally highly related to the RNase P particle, which is involved in pre-tRNA processing. Their RNA components fold into a similar secondary structure and they share several protein subunits. We have identified and characterised human and mouse cDNAs that encode proteins homologous to yPop4p, a protein subunit of both the yeast RNase MRP and RNase P complexes. The human Pop4 cDNA encodes a highly basic protein of 220 amino acids. Transfection experiments with epitope-tagged hPop4 protein indicated that hPop4 is localised in the nucleus and accumulates in the nucleolus. Immunoprecipitation assays using extracts from transfected cells expressing epitope-tagged hPop4 revealed that this protein is associated with both the human RNase MRP and RNase P particles. Polyclonal rabbit antibodies raised against recombinant hPop4 recognised a 30 kDa protein in total HeLa cell extracts and specifically co-immunoprecipitated the RNA components of the RNase MRP and RNase P complexes. Finally we showed that anti-hPop4 immunoprecipitates possess RNase P enzymatic activity. Taken together, these data show that we have identified a protein that represents the human counterpart of the yeast Pop4p protein.     

Footprinting analysis demonstrates extensive similarity between eukaryotic RNase P and RNase MRP holoenzymes

Eukaryotic ribonuclease (RNase) P and RNase MRP are evolutionary related RNA-based enzymes involved in metabolism of various RNA molecules, including tRNA and rRNA. In contrast to the closely related eubacterial RNase P, which is comprised of an RNA component and a single small protein, these enzymes contain multiple protein components. Here we report the results of footprinting studies performed on purified Saccharomyces cerevisiae RNase MRP and RNase P holoenzymes. The results identify regions of the RNA components affected by the protein moiety, suggest a role of the proteins in stabilization of the RNA fold, and point to substantial similarities between the two evolutionary related RNA-based enzymes.     

Secondary structure probing of the human RNase MRP RNA reveals the potential for MRP RNA subsets

RNase MRP is a ribonucleoprotein endoribonuclease involved in eukaryotic pre-rRNA processing. The enzyme possesses an RNA subunit, structurally related to that of RNase P RNA, that is thought to be catalytic. RNase MRP RNA sequences from Saccharomycetaceae species are structurally well defined through detailed phylogenetic and structural analysis. In contrast, higher eukaryote MRP RNA structure models are based on comparative sequence analysis of only five sequences and limited probing data. Detailed structural analysis of the Homo sapiens MRP RNA, entailing enzymatic and chemical probing, is reported. The data are consistent with the phylogenetic secondary structure model and demonstrate unequivocally that higher eukaryote MRP RNA structure differs significantly from that reported for Saccharomycetaceae species. Neither model can account for all of the known MRP RNAs and we thus propose the evolution of at least two subsets of RNase MRP secondary structure, differing predominantly in the predicted specificity domain.     

Mutual interactions between subunits of the human RNase MRP ribonucleoprotein complex

The eukaryotic ribonuclease for mitochondrial RNA processing (RNase MRP) is mainly located in the nucleoli and belongs to the small nucleolar ribonucleoprotein (snoRNP) particles. RNase MRP is involved in the processing of pre-rRNA and the generation of RNA primers for mitochondrial DNA replication. A closely related snoRNP, which shares protein subunits with RNase MRP and contains a structurally related RNA subunit, is the pre-tRNA processing factor RNase P. Up to now, 10 protein subunits of these complexes have been described, designated hPop1, hPop4, hPop5, Rpp14, Rpp20, Rpp21, Rpp25, Rpp30, Rpp38 and Rpp40. To get more insight into the assembly of the human RNase MRP complex we studied protein–protein and protein–RNA interactions by means of GST pull-down experiments. A total of 19 direct protein–protein and six direct protein–RNA interactions were observed. The analysis of mutant RNase MRP RNAs showed that distinct regions are involved in the direct interaction with protein subunits. The results provide insight into the way the protein and RNA subunits assemble into a ribonucleoprotein particle. Based upon these data a new model for the architecture of the human RNase MRP complex was generated.     

Genetic and biochemical analyses of yeast RNase MRP

RNase MRP cleaves the yeast pre-rRNA at a site in internal transcribed spacer 1 (ITS1) and this cleavage can be reproducedin vitro by the highly purified enzyme. Two protein components (Pop1p and Pop2p) have been identified which are common to yeast RNase MRP and RNase P. Moreover, purified RNase P can also cleave the pre-rRNA substratein vitro, underlining the similarities between these particles. Genetic evidence suggests that RNase MRP functionally interacts with the snoRNPs which are required for other pre-rRNA processing reactions.Abbreviations pre-rRNA ribosomal RNA precursor - snoRNA small nucleolar RNA - snoRNP small nucleolar ribonucleoprotein particle     

RNase MRP and rRNA processing

RNase MRP is a ribonucleoprotein enzyme with a structure similar to RNase P. It is required for normal processing of precursor rRNA, cleaving it in the Internal Transcribed Spacer 1. Abbreviations: RNase MRP RNase for mitochondrial RNA processing; also involved in pre-rRNA processing; RNase P - RNase for pre-tRNA processing; snoRNA - small nucleolar RNA; RNP - RNA-protein particle; snoRNP - small nucleolar RNA-protein particle.     

Autoantigenic properties of some protein subunits of catalytically active complexes of human ribonuclease P.

At least six proteins co-purify with human ribonuclease P (RNase P), a tRNA processing ribonucleoprotein. Two of these proteins, Rpp30 and Rpp38, are Th autoantigens. Recombinant Rpp30 and Rpp38 are also recognized by Th sera from systemic sclerosis patients. Two of the other proteins associated with RNase P, Rpp20 and Rpp40, do not cross-react with Th sera. Polyclonal antibodies raised against all four recombinant proteins recognize the corresponding proteins associated with RNase P and precipitate active holoenzyme. Catalytically active RNase P holoenzyme can be separated from the nucleolar and mitochondrial RNA processing endoribonuclease, RNase MRP, even though these two enzymes may share some subunits.