Alterations in the intracellular level of a protein subunit of human RNase P affect processing of tRNA precursors

The human ribonucleoprotein ribonuclease P (RNase P), processing tRNA, has at least 10 distinct protein subunits. Many of these subunits, including the autoimmune antigen Rpp38, are shared by RNase MRP, a ribonucleoprotein enzyme required for processing of rRNA. We here show that constitutive expression of exogenous, tagged Rpp38 protein in HeLa cells affects processing of tRNA precursors. Alterations in the site-specific cleavage and in the steady-state level of 3′ sequences of the internal transcribed spacer 1 of rRNA are also observed. These processing defects are accompanied by selective shut-off of expression of Rpp38 and by low expression of the tagged protein. RNase P purified from these cells exhibits impaired activity in vitro. Moreover, inhibition of Rpp38 by the use of small interfering RNA causes accumulation of the initiator methionine tRNA precursor. Expression of other protein components, but not of the H1 RNA subunit, is coordinately inhibited. Our results reveal that normal expression of Rpp38 is required for the biosynthesis of intact RNase P and for the normal processing of stable RNA in human cells.     

Basic Domains Target Protein Subunits of the RNase MRP Complex to the Nucleolus Independently of Complex Association

The RNase MRP and RNase P ribonucleoprotein particles both function as endoribonucleases, have a similar RNA component, and share several protein subunits. RNase MRP has been implicated in pre-rRNA processing and mitochondrial DNA replication, whereas RNase P functions in pre-tRNA processing. Both RNase MRP and RNase P accumulate in the nucleolus of eukaryotic cells. In this report we show that for three protein subunits of the RNase MRP complex (hPop1, hPop4, and Rpp38) basic domains are responsible for their nucleolar accumulation and that they are able to accumulate in the nucleolus independently of their association with the RNase MRP and RNase P complexes. We also show that certain mutants of hPop4 accumulate in the Cajal bodies, suggesting that hPop4 traverses through these bodies to the nucleolus. Furthermore, we characterized a deletion mutant of Rpp38 that preferentially associates with the RNase MRP complex, giving a first clue about the difference in protein composition of the human RNase MRP and RNase P complexes. On the basis of all available data on nucleolar localization sequences, we hypothesize that nucleolar accumulation of proteins containing basic domains proceeds by diffusion and retention rather than by an active transport process. The existence of nucleolar localization sequences is discussed.     

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.     

Rpp14 and Rpp29, two protein subunits of human ribonuclease P

In HeLa cells, the tRNA processing enzyme ribonuclease P (RNase P) consists of an RNA molecule associated with at least eight protein subunits, hPop1, Rpp14, Rpp20, Rpp25, Rpp29, Rpp30, Rpp38, and Rpp40. Five of these proteins (hPop1p, Rpp20, Rpp30, Rpp38, and Rpp40) have been partially characterized. Here we report on the cDNA cloning and immunobiochemical analysis of Rpp14 and Rpp29. Polyclonal rabbit antibodies raised against recombinant Rpp14 and Rpp29 recognize their corresponding antigens in HeLa cells and precipitate catalytically active RNase P. Rpp29 shows 23% identity with Pop4p, a subunit of yeast nuclear RNase P and the ribosomal RNA processing enzyme RNase MRP. Rpp14, by contrast, exhibits no significant homology to any known yeast gene. Thus, human RNase P differs in the details of its protein composition, and perhaps in the functions of some of these proteins, from the yeast enzyme.     

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.     

Differential association of protein subunits with the human RNase MRP and RNase P complexes

RNase MRP is a eukaryotic endoribonuclease involved in nucleolar and mitochondrial RNA processing events. RNase MRP is a ribonucleoprotein particle, which is structurally related to RNase P, an endoribonuclease involved in pre-tRNA processing. Most of the protein components of RNase MRP have been reported to be associated with RNase P as well. In this study we determined the association of these protein subunits with the human RNase MRP and RNase P particles by glycerol gradient sedimentation and coimmunoprecipitation. In agreement with previous studies, RNase MRP sedimented at 12S and 60-80S. In contrast, only a single major peak was observed for RNase P at 12S. The analysis of individual protein subunits revealed that hPop4 (also known as Rpp29), Rpp21, Rpp20, and Rpp25 only sedimented in 12S fractions, whereas hPop1, Rpp40, Rpp38, and Rpp30 were also found in 60-80S fractions. In agreement with their cosedimentation with RNase P RNA in the 12S peak, coimmunoprecipitation with VSV-epitope-tagged protein subunits revealed that hPop4, Rpp21, and in addition Rpp14 preferentially associate with RNase P. These data show that hPop4, Rpp21, and Rpp14 may not be associated with RNase MRP. Furthermore, Rpp20 and Rpp25 appear to be associated with only a subset of RNase MRP particles, in contrast to hPop1, Rpp40, Rpp38, and Rpp30 (and possibly also hPop5), which are probably associated with all RNase MRP complexes. Our data are consistent with a transient association of Rpp20 and Rpp25 with RNase MRP, which may be inversely correlated to its involvement in pre-rRNA processing.     

Purification and characterization of Rpp25, an RNA-binding protein subunit of human ribonuclease P

In HeLa cells, ribonuclease P (RNase P), the tRNA processing enzyme consists of an RNA subunit (H1 RNA) associated with at least nine protein subunits, Rpp14, Rpp20, Rpp21, Rpp29 (hPop4), Rpp30, Rpp38, Rpp40, hPop1, and hPop5 (18.8 kDa). We report here the cloning and immuno-biochemical analysis of Rpp25, another protein subunit of RNase P. Polyclonal rabbit antibodies raised against recombinant Rpp25 recognize their corresponding antigens in RNase P-containing fractions purified from HeLa cells, and they also precipitate active holoenzyme. Furthermore, this protein has general RNA binding properties.     

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.     

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.     

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.     

Structure of an archaeal homolog of the human protein complex Rpp21-Rpp29 that is a key core component for the assembly of active ribonuclease P

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5′-leader sequence of precursor tRNA. Human RNase P protein subunits Rpp21 and Rpp29, which bind to each other, with catalytic RNA (H1 RNA) are sufficient for activating endonucleolytic cleavage of precursor tRNA. Here we have determined the crystal structure of the complex between the Pyrococcus horikoshii RNase P proteins PhoRpp21 and PhoRpp29, the archaeal homologs of Rpp21 and Rpp29, respectively. PhoRpp21 and PhoRpp29 form a heterodimeric structure where the two N-terminal helices (α1 and α2) in PhoRpp21 predominantly interact with the N-terminal extended structure, the β-strand (β2), and the C-terminal helix (α3) in PhoRpp29. The interface is dominated by hydrogen bonds and several salt bridges, rather than hydrophobic interactions. The electrostatic potential on the surface of the heterodimer shows a positively charged cluster on one face, suggesting a possible RNA-binding surface of the PhoRpp21-PhoRpp29 complex. The present structure, along with the result of a mutational analysis, suggests that heterodimerization between PhoRpp21 and PhoRpp29 plays an important role in the function of P. horikoshii RNase P.     

Crystal Structure of Human Rpp20/Rpp25 Reveals Quaternary Level Adaptation of the Alba Scaffold as Structural Basis for Single-stranded RNA Binding

Ribonuclease P (RNase P) catalyzes the removal of 5′ leaders of tRNA precursors and its central catalytic RNA subunit is highly conserved across all domains of life. In eukaryotes, RNase P and RNase MRP, a closely related ribonucleoprotein enzyme, share several of the same protein subunits, contain a similar catalytic RNA core, and exhibit structural features that do not exist in their bacterial or archaeal counterparts. A unique feature of eukaryotic RNase P/MRP is the presence of two relatively long and unpaired internal loops within the P3 region of their RNA subunit bound by a heterodimeric protein complex, Rpp20/Rpp25. Here we present a crystal structure of the human Rpp20/Rpp25 heterodimer and we propose, using comparative structural analyses, that the evolutionary divergence of the single-stranded and helical nucleic acid binding specificities of eukaryotic Rpp20/Rpp25 and their related archaeal Alba chromatin protein dimers, respectively, originate primarily from quaternary level differences observed in their heterodimerization interface. Our work provides structural insights into how the archaeal Alba protein scaffold was adapted evolutionarily for incorporation into several functionally-independent eukaryotic ribonucleoprotein complexes.     

RNase P: role of distinct protein cofactors in tRNA substrate recognition and RNA-based catalysis

The Escherichia coli ribonuclease P (RNase P) has a protein component, termed C5, which acts as a cofactor for the catalytic M1 RNA subunit that processes the 5′ leader sequence of precursor tRNA. Rpp29, a conserved protein subunit of human RNase P, can substitute for C5 protein in reconstitution assays of M1 RNA activity. To better understand the role of the former protein, we compare the mode of action of Rpp29 to that of the C5 protein in activation of M1 RNA. Enzyme kinetic analyses reveal that complexes of M1 RNA–Rpp29 and M1 RNA–C5 exhibit comparable binding affinities to precursor tRNA but different catalytic efficiencies. High concentrations of substrate impede the activity of the former complex. Rpp29 itself exhibits high affinity in substrate binding, which seems to reduce the catalytic efficiency of the reconstituted ribonucleoprotein. Rpp29 has a conserved C-terminal domain with an Sm-like fold that mediates interaction with M1 RNA and precursor tRNA and can activate M1 RNA. The results suggest that distinct protein folds in two unrelated protein cofactors can facilitate transition from RNA- to ribonucleoprotein-based catalysis by RNase P.     

Inventory and analysis of the protein subunits of the ribonucleases P and MRP provides further evidence of homology between the yeast and human enzymes

The RNases P and MRP are involved in tRNA and rRNA processing, respectively. Both enzymes in eukaryotes are composed of an RNA molecule and 9–12 protein subunits. Most of the protein subunits are shared between RNases P and MRP. We have here performed a computational analysis of the protein subunits in a broad range of eukaryotic organisms using profile-based searches and phylogenetic methods. A number of novel homologues were identified, giving rise to a more complete inventory of RNase P/MRP proteins. We present evidence of a relationship between fungal Pop8 and the protein subunit families Rpp14/Pop5 as well as between fungal Pop6 and metazoan Rpp25. These relationships further emphasize a structural and functional similarity between the yeast and human P/MRP complexes. We have also identified novel P and MRP RNAs and analysis of all available sequences revealed a K-turn motif in a large number of these RNAs. We suggest that this motif is a binding site for the Pop3/Rpp38 proteins and we discuss other structural features of the RNA subunit and possible relationships to the protein subunit repertoire.     

Protein-RNA interactions in the subunits of human nuclear RNase P.

A yeast three-hybrid system was employed to analyze interactions in vivo between H1 RNA, the RNA subunit of human nuclear RNase P, and eight of the protein subunits of the enzyme. The genetic analysis indicates that subunits Rpp21, Rpp29, Rpp30, and Rpp38 interact directly with H1 RNA. The results of direct UV crosslinking studies of the purified RNase P holoenzyme confirm the results of the three-hybrid assay.     

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.     

Heterodimerization regulates RNase MRP/RNase P association, localization, and expression of Rpp20 and Rpp25

Rpp20 and Rpp25 are subunits of the human RNase MRP and RNase P endoribonucleases belonging to the Alba superfamily of nucleic acid binding proteins. These proteins, which bind very strongly to each other, transiently associate with RNase MRP. Here, we show that the Rpp20-Rpp25 heterodimer is resistant to both high concentrations of salt and a nonionic detergent. The interaction of Rpp20 and Rpp25 with the P3 domain of the RNase MRP RNA appeared to be strongly enhanced by their heterodimerization. Coimmunoprecipitation experiments demonstrated that only a single copy of each of these proteins is associated with the RNase MRP and RNase P particles in HEp-2 cells. Both proteins accumulate in the nucleoli, which in case of Rpp20 is strongly dependent on its interaction with Rpp25. Finally, the results of overexpression and knock-down experiments indicate that their expression levels are codependent. Taken together, these data indicate that the Rpp20-Rpp25 heterodimerization regulates their RNA-binding activity, subcellular localization, and expression, which suggests that their interaction is also crucial for their role in RNase MRP/P function.     

Structural basis for activation of an archaeal ribonuclease P RNA by protein cofactors

Ribonuclease P (RNase P) is an endoribonuclease that catalyzes the processing of the 5′-leader sequence of precursor tRNA (pre-tRNA) in all phylogenetic domains. We have found that RNase P in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 consists of RNase P RNA (PhopRNA) and five protein cofactors designated PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38. Biochemical characterizations over the past 10 years have revealed that PhoPop5 and PhoRpp30 fold into a heterotetramer and cooperate to activate a catalytic domain (C-domain) in PhopRNA, whereas PhoRpp21 and PhoRpp29 form a heterodimer and function together to activate a specificity domain (S-domain) in PhopRNA. PhoRpp38 plays a role in elevation of the optimum temperature of RNase P activity, binding to kink-turn (K-turn) motifs in two stem-loops in PhopRNA. This review describes the structural and functional information on P. horikoshii RNase P, focusing on the structural basis for the PhopRNA activation by the five RNase P proteins.