Phylogenetic analysis of the structure of RNase MRP RNA in yeasts

RNase MRP is a ribonucleoprotein enzyme involved in processing precursor rRNA in eukaryotes. To facilitate our structure-function analysis of RNase MRP from Saccharomyces cerevisiae, we have determined the likely secondary structure of the RNA component by a phylogenetic approach in which we sequenced all or part of the RNase MRP RNAs from 17 additional species of the Saccharomycetaceae family. The structure deduced from these sequences contains the helices previously suggested to be common to the RNA subunit of RNase MRP and the related RNA subunit of RNase P, an enzyme cleaving tRNA precursors. However, outside this common region, the structure of RNase MRP RNA determined here differs from a previously proposed universal structure for RNase MRPs. Chemical and enzymatic structure probing analyses were consistent with our revised secondary structure. Comparison of all known RNase MRP RNA sequences revealed three regions with highly conserved nucleotides. Two of these regions are part of a helix implicated in RNA catalysis in RNase P, suggesting that RNase MRP may cleave rRNA using a similar catalytic mechanism.     

A conserved element in the yeast RNase MRP RNA subunit can participate in a long-range base-pairing interaction

RNase MRP is a ribonucleoprotein endoribonuclease involved in eukaryotic pre-rRNA processing. The enzyme possesses a putatively catalytic RNA subunit, structurally related to that of RNase P. A thorough structure analysis of Saccharomyces cerevisiae MRP RNA, entailing enzymatic and chemical probing, mutagenesis and thermal melting, identifies a previously unrecognised stem that occupies a position equivalent to the P7 stem of RNase P. Inclusion of this P7-like stem confers on yeast MRP RNA a greater degree of similarity to the core RNase P RNA structure than that described previously and better delimits domain 2, the proposed specificity domain. The additional stem is created by participation of a conserved sequence element (ymCR-II) in a long-range base-pairing interaction. There is potential for this base-pairing throughout the known yeast MRP RNA sequences. Formation of a P7-like stem is not required, however, for the pre-rRNA processing or essential function of RNase MRP. Mutants that can base-pair are nonetheless detrimental to RNase MRP function, indicating that the stem will form in vivo but that only the wild-type pairing is accommodated. Although the alternative MRP RNA structure described is clearly not part of the active RNase MRP enzyme, it would be the more stable structure in the absence of protein subunits and the probability that it represents a valid intermediate species in the process of yeast RNase MRP assembly is discussed.     

Use of RNA Secondary Structure for Studying the Evolution of RNase P and RNase MRP

Secondary structure is evaluated for determining evolutionary relationships between catalytic RNA molecules that are so distantly related they are scarcely alignable. The ribonucleoproteins RNase P (P) and RNase MRP (MRP) have been suggested to be evolutionarily related because of similarities in both function and secondary structure. However, their RNA sequences cannot be aligned with any confidence, and this leads to uncertainty in any trees inferred from sequences. We report several approaches to using secondary structures for inferring evolutionary trees and emphasize quantitative tests to demonstrate that evolutionary information can be recovered. For P and MRP, three hypotheses for the relatedness are considered. The first is that MRP is derived from P in early eukaryotes. The next is that MRP is derived from P from an early endosymbiont. The third is that both P and MRP evolved in the RNA-world (and the need for MRP has since been lost in prokaryotes). Quantitative comparisons of the pRNA and mrpRNA secondary structures have found that the possibility of an organellar origin of MRP is unlikely. In addition, comparison of secondary structures support the identity of an RNase P–like sequence in the maize chloroplast genome. Overall, it is concluded that RNA secondary structure is useful for evaluating evolutionary relatedness, even with sequences that cannot be aligned with confidence. Received: 19 July 1999 / Accepted: 3 May 2000     

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.     

RNase MRP RNA and human genetic diseases

RNase MRP RNA is the RNA subunit of the RNase mitochondrial RNA processing （MRP） enzyme complex that is involved in multiple cellular RNA processing events. Mutations on RNase MRP RNA gene （RMRP） cause a recessively inherited developmental disorder, cartilage-hair hypoplasia （CHH）. The relationship of the genotype （RMRP mutation）, RNA processing deficiency of the RNase MRP complex, and the phenotype of CHH and other skeletal dysplasias is yet to be explored.     

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.     

Identification and analysis of ribonuclease P and MRP RNA in a broad range of eukaryotes

RNases P and MRP are ribonucleoprotein complexes involved in tRNA and rRNA processing, respectively. The RNA subunits of these two enzymes are structurally related to each other and play an essential role in the enzymatic reaction. Both of the RNAs have a highly conserved helical region, P4, which is important in the catalytic reaction. We have used a bioinformatics approach based on conserved elements to computationally analyze available genomic sequences of eukaryotic organisms and have identified a large number of novel nuclear RNase P and MRP RNA genes. For MRP RNA for instance, this investigation increases the number of known sequences by a factor of three. We present secondary structure models of many of the predicted RNAs. Although all sequences are able to fold into the consensus secondary structure of P and MRP RNAs, a striking variation in size is observed, ranging from a Nosema locustae MRP RNA of 160 nt to much larger RNAs, e.g. a Plasmodium knowlesi P RNA of 696 nt. The P and MRP RNA genes appear in tandem in some protists, further emphasizing the close evolutionary relationship of these RNAs.     

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.     

The evolution of the RNase P- and RNase MRP-associated RNAs: Phylogenetic analysis and nucleotide substitution rate

We report a detailed evolutionary study of the RNase P- and RNase MRP- associated RNAs. The analyses were performed on all the available complete sequences of RNase MRP (vertebrates, yeast, plant), nuclear RNase P (vertebrates, yeast), and mitochondrial RNase P (yeast) RNAs. For the first time the phylogenetic distance between these sequences and the nucleotide substitution rates have been quantitatively measured. The analyses were performed by considering the optimal multiple alignments obtained mostly by maximizing similarity between primary sequences. RNase P RNA and MRP RNA display evolutionary dynamics following the molecular clock. Both have similar rates and evolve about one order of magnitude faster than the corresponding small rRNA sequences which have been, so far, the most common gene markers used for phylogeny. However, small rRNAs evolve too slowly to solve close phylogenetic relationships such as those between mammals. The quicker rate of RNase P and MRP RNA allowed us to assess phylogenetic relationships between mammals and other vertebrate species and yeast strains. The phylogenetic data obtained with yeasts perfectly agree with those obtained by functional assays, thus demonstrating the potential offered by this approach for laboratory experiments. Correspondence to: E. Sbisà     

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.     

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.     

Eukaryotic ribonucleases P/MRP: the crystal structure of the P3 domain

Ribonuclease (RNase) P is a site‐specific endoribonuclease found in all kingdoms of life. Typical RNase P consists of a catalytic RNA component and a protein moiety. In the eukaryotes, the RNase P lineage has split into two, giving rise to a closely related enzyme, RNase MRP, which has similar components but has evolved to have different specificities. The eukaryotic RNases P/MRP have acquired an essential helix‐loop‐helix protein‐binding RNA domain P3 that has an important function in eukaryotic enzymes and distinguishes them from bacterial and archaeal RNases P. Here, we present a crystal structure of the P3 RNA domain from Saccharomyces cerevisiae RNase MRP in a complex with RNase P/MRP proteins Pop6 and Pop7 solved to 2.7 Å. The structure suggests similar structural organization of the P3 RNA domains in RNases P/MRP and possible functions of the P3 domains and proteins bound to them in the stabilization of the holoenzymes' structures as well as in interactions with substrates. It provides the first insight into the structural organization of the eukaryotic enzymes of the RNase P/MRP family.     

Substrate recognition by ribonucleoprotein ribonuclease MRP

The ribonucleoprotein complex ribonuclease (RNase) MRP is a site-specific endoribonuclease essential for the survival of the eukaryotic cell. RNase MRP closely resembles RNase P (a universal endoribonuclease responsible for the maturation of the 5' ends of tRNA) but recognizes distinct substrates including pre-rRNA and mRNA. Here we report the results of an in vitro selection of Saccharomyces cerevisiae RNase MRP substrates starting from a pool of random sequences. The results indicate that RNase MRP cleaves single-stranded RNA and is sensitive to sequences in the immediate vicinity of the cleavage site requiring a cytosine at the position +4 relative to the cleavage site. Structural implications of the differences in substrate recognition by RNases P and MRP are discussed.     

Efficient site-specific cleavage by RNase MRP requires interaction with two evolutionarily conserved mitochondrial RNA sequences.

RNase MRP is a site-specific endonuclease that processes primer mitochondrial RNA from the leading-strand origin of mitochondrial DNA replication. Using deletional analysis and saturation mutagenesis, we have determined the substrate requirements for cleavage by mouse mitochondrial RNase MRP. Two regions of sequence homology among vertebrate mitochondrial RNA primers, conserved sequence blocks II and III, were found to be critical for both efficient and accurate cleavage; a third region of sequence homology, conserved sequence block I, was dispensable. Analysis of insertion and deletion mutations within conserved sequence block II demonstrated that the specificity of RNase MRP accommodates the natural sequence heterogeneity of conserved sequence block II in vivo. Heterologous assays with human RNase MRP and mutated mouse mitochondrial RNA substrates indicated that sequences essential for substrate recognition are conserved between mammalian species.     

Bovine RNase MRP cleaves the divergent bovine mitochondrial RNA sequence at the displacement-loop region

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves mitochondrial RNA from the origin of leading-strand DNA synthesis contained within the displacement-loop region. Bovine mitochondrial DNA maintains the typical gene content and order of mammalian mitochondrial DNAs but differs in the nature of sequence conservation within this displacement-loop regulatory region. This markedly different sequence arrangement raises the issue of the degree to which a bovine RNase MRP would reflect the physical and functional properties ascribed to the enzymes previously characterized from mouse and human. We find that bovine RNase MRP exists as a ribonucleoprotein, with an RNA component of 279 nucleotides that is homologous to that of mouse or human RNase MRP RNA. Characterization of the nuclear gene for bovine RNase MRP RNA showed conservation of sequence extending 5 of the RNase MRP RNA coding sequence, including the presence of a cis-acting element known to be important for the expression of some mitochondrial protein-coding nuclear genes. Bovine or mouse RNase MRP cleaves a standard mouse mitochondrial RNA substrate in the same manner; each also cleaves a bovine mitochondrial RNA substrate identically. Since bovine and mouse RNase MRPs process both bovine and mouse substrates, we conclude that the structural features of the mitochondrial RNA substrate required for enzymatic cleavage have been well conserved despite significant overall primary sequence divergence. Inspection of the bovine RNA substrate reveals conservation of only the most critical portion of the primary sequence as indicated by earlier studies with mouse and human RNase MRPs. Interestingly, a principal cleavage site in the bovine mitochondrial RNA substrate is downstream of the promoter located at the leading-strand mitochondrial DNA replication origin. Correspondence to: D.J. Dairaghi     

RNase MRP and the RNA processing cascade in the eukaryotic ancestor

Background

Within eukaryotes there is a complex cascade of RNA-based macromolecules that process other RNA molecules, especially mRNA, tRNA and rRNA. An example is RNase MRP processing ribosomal RNA (rRNA) in ribosome biogenesis. One hypothesis is that this complexity was present early in eukaryotic evolution; an alternative is that an initial simpler network later gained complexity by gene duplication in lineages that led to animals, fungi and plants. Recently there has been a rapid increase in support for the complexity-early theory because the vast majority of these RNA-processing reactions are found throughout eukaryotes, and thus were likely to be present in the last common ancestor of living eukaryotes, herein called the Eukaryotic Ancestor.

Results

We present an overview of the RNA processing cascade in the Eukaryotic Ancestor and investigate in particular, RNase MRP which was previously thought to have evolved later in eukaryotes due to its apparent limited distribution in fungi and animals and plants. Recent publications, as well as our own genomic searches, find previously unknown RNase MRP RNAs, indicating that RNase MRP has a wide distribution in eukaryotes. Combining secondary structure and promoter region analysis of RNAs for RNase MRP, along with analysis of the target substrate (rRNA), allows us to discuss this distribution in the light of eukaryotic evolution.

Conclusion

We conclude that RNase MRP can now be placed in the RNA-processing cascade of the Eukaryotic Ancestor, highlighting the complexity of RNA-processing in early eukaryotes. Promoter analyses of MRP-RNA suggest that regulation of the critical processes of rRNA cleavage can vary, showing that even these key cellular processes (for which we expect high conservation) show some species-specific variability. We present our consensus MRP-RNA secondary structure as a useful model for further searches.

     

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.