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.     

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.     

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     

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.     

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.     

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.     

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.     

Analysis of the tertiary structure of bacterial RNase P RNA

The ubiquitous occurrence of ribonuclease P (RNase P) as a ribonucleoprotein and the catalytic properties of bacterial RNase P RNAs indicate that RNA fulfills an ancient and important role in the function of this enzyme. This review focuses on efforts to determine the structure of the bacterial RNase P RNA ribozyme. Phylogenetic comparative analysis of a library of bacterial RNase P RNA sequences has resulted in a well-developed secondary structure model and allowed identification of some elements of tertiary structure. The native structure has been redesigned by circular permutation to facilitate intra- and inter-molecular crosslinking experiments in order to gain further structural information. The crosslinking constraints, together with the constraints provided by comparative analyses, have been incorporated into a first-order model of the structure of the ribozyme-substrate complex. The developing structural perspective allows the design of self-cleaving pre-tRNA-RNase P RNA conjugates which are useful tools for additional structure-probing experiments.Abbreviations cpRNA circularly permuted RNA     

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.     

Structure-function analysis in nuclear RNase P RNA

Eukaryotic ribonuclease P (RNase P) enzymes require both RNA and protein subunits for activityin vivo andin vitro. We have undertaken an analysis of the complex RNA subunit of the nuclear holoenzyme in an effort to understand its structure and its similarities to and differences from the bacterial ribozymes. Phylogenetic analysis, structure-sensitive RNA footprinting, and directed mutagenesis reveal conserved secondary and tertiary structures with both strong similarities to the bacterial consensus and distinctive features. The effects of mutations in the most highly conserved positions are being used to dissect the functions of individual subdomains.Abbreviations RPRI ribonucleaseP ribonucleoprotein 1 gene fromSaccharomyces cerevisiae - Pu purine ribonucleoside     

Comparison of mitochondrial and nucleolar RNase MRP reveals identical RNA components with distinct enzymatic activities and protein components

RNase MRP is a ribonucleoprotein endoribonuclease found in three cellular locations where distinct substrates are processed: the mitochondria, the nucleolus, and the cytoplasm. Cytoplasmic RNase MRP is the nucleolar enzyme that is transiently relocalized during mitosis. Nucleolar RNase MRP (NuMRP) was purified to homogeneity, and we extensively purified the mitochondrial RNase MRP (MtMRP) to a single RNA component identical to the NuMRP RNA. Although the protein components of the NuMRP were identified by mass spectrometry successfully, none of the known NuMRP proteins were found in the MtMRP preparation. Only trace amounts of the core NuMRP protein, Pop4, were detected in MtMRP by Western blot. In vitro activity of the two enzymes was compared. MtMRP cleaved only mitochondrial ORI5 substrate, while NuMRP cleaved all three substrates. However, the NuMRP enzyme cleaved the ORI5 substrate at sites different than the MtMRP enzyme. In addition, enzymatic differences in preferred ionic strength confirm these enzymes as distinct entities. Magnesium was found to be essential to both enzymes. We tested a number of reported inhibitors including puromycin, pentamidine, lithium, and pAp. Puromycin inhibition suggested that it binds directly to the MRP RNA, reaffirming the role of the RNA component in catalysis. In conclusion, our study confirms that the NuMRP and MtMRP enzymes are distinct entities with differing activities and protein components but a common RNA subunit, suggesting that the RNA must be playing a crucial role in catalytic activity.     

Solution structure of RNase P RNA

The ribonucleoprotein enzyme ribonuclease P (RNase P) processes tRNAs by cleavage of precursor-tRNAs. RNase P is a ribozyme: The RNA component catalyzes tRNA maturation in vitro without proteins. Remarkable features of RNase P include multiple turnovers in vivo and ability to process diverse substrates. Structures of the bacterial RNase P, including full-length RNAs and a ternary complex with substrate, have been determined by X-ray crystallography. However, crystal structures of free RNA are significantly different from the ternary complex, and the solution structure of the RNA is unknown. Here, we report solution structures of three phylogenetically distinct bacterial RNase P RNAs from Escherichia coli, Agrobacterium tumefaciens, and Bacillus stearothermophilus, determined using small angle X-ray scattering (SAXS) and selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis. A combination of homology modeling, normal mode analysis, and molecular dynamics was used to refine the structural models against the empirical data of these RNAs in solution under the high ionic strength required for catalytic activity.     

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     

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