Mutational analysis of the RNA component of Saccharomyces cerevisiae RNase MRP reveals distinct nuclear phenotypes

The 340-nucleotide RNA component of Saccharomyces cerevisiae RNase MRP is encoded by the single-copy essential gene, NME1. To gain additional insight into the proposed structure and functions of this endoribonuclease, we have extensively mutagenized the NME1 gene and characterized yeast strains expressing mutated forms of the RNA using a gene shuffle technique. Strains expressing each of 26 independent mutations in the RNase MRP RNA gene were characterized for their ability to grow at various temperatures and on various carbon sources, stability of the RNase MRP RNA and processing of the 5.8S rRNA (a nuclear function of RNase MRP). 11 of the mutations resulted in a lethal phenotype, six displayed temperature-conditional lethality, and several preferred a non-fermentable carbon source for growth. In those mutants that exhibited altered growth phenotypes, the severity of the growth defect was directly proportional to the severity of the 5.8S rRNA processing defect in the nucleus. Together this analysis has defined essential regions of the RNase MRP RNA and provides evidence that is consistent with the proposed function of the RNase MRP enzyme.     

The RNA catabolic enzymes Rex4p, Rnt1p, and Dbr1p show genetic interaction with trans-acting factors involved in processing of ITS1 in Saccharomyces cerevisiae pre-rRNA

Eukaryotes have two types of ribosomes containing either 5.8SL or 5.8SS rRNA that are produced by alternative pre-rRNA processing. The exact processing pathway for the minor 5.8SL rRNA species is poorly documented. We have previously shown that the trans-acting factor Rrp5p and the RNA exonuclease Rex4p genetically interact to influence the ratio between the two forms of 5.8S rRNA in the yeast Saccharomyces cerevisiae. Here we report a further analysis of ITS1 processing in various yeast mutants that reveals genetic interactions between, on the one hand, Rrp5p and RNase MRP, the endonuclease required for 5.8SS rRNA synthesis, and, on the other, Rex4p, the RNase III homolog Rnt1p, and the debranching enzyme Dbr1p. Yeast cells carrying a temperature-sensitive mutation in RNase MRP (rrp2-1) exhibit a pre-rRNA processing phenotype very similar to that of the previously studied rrp5-33 mutant: ITS2 processing precedes ITS1 processing, 5.8SL rRNA becomes the major species, and ITS1 is processed at the recently reported novel site A4 located midway between sites A2 and A3. As in the rrp5-Delta3 mutant, all of these phenotypical processing features disappear upon inactivation of the REX4 gene. Moreover, inactivation of the DBR1 gene in rrp2-1, or the RNT1 gene in rrp5-Delta3 mutant cells also negates the effects of the original mutation on pre-rRNA processing. These data link a total of three RNA catabolic enzymes, Rex4p, Rnt1p, and Dbr1p, to ITS1 processing and the relative production of 5.8SS and 5.8SL rRNA. A possible model for the indirect involvement of the three enzymes in yeast pre-rRNA processing is discussed.     

Mutagenesis of SNM1, Which Encodes a Protein Component of the Yeast RNase MRP, Reveals a Role for This Ribonucleoprotein Endoribonuclease in Plasmid Segregation

RNase MRP is a ribonucleoprotein endoribonuclease that has been shown to have roles in both mitochondrial DNA replication and nuclear 5.8S rRNA processing. SNM1 encodes an essential 22.5-kDa protein that is a component of yeast RNase MRP. It is an RNA binding protein that binds the MRP RNA specifically. This 198-amino-acid protein can be divided into three structural regions: a potential leucine zipper near the amino terminus, a binuclear zinc cluster in the middle region, and a serine- and lysine-rich region near the carboxy terminus. We have performed PCR mutagenesis of the SNM1 gene to produce 17 mutants that have a conditional phenotype for growth at different temperatures. Yeast strains carrying any of these mutations as the only copy of snm1 display an rRNA processing defect identical to that in MRP RNA mutants. We have characterized these mutant proteins for RNase MRP function by examining 5.8S rRNA processing, MRP RNA binding in vivo, and the stability of the RNase MRP RNA. The results indicate two separate functional domains of the protein, one responsible for binding the MRP RNA and a second that promotes substrate cleavage. The Snm1 protein appears not to be required for the stability of the MRP RNA, but very low levels of the protein are required for processing of the 5.8S rRNA. Surprisingly, a large number of conditional mutations that resulted from nonsense and frameshift mutations throughout the coding regions were identified. The most severe of these was a frameshift at amino acid 7. These mutations were found to be undergoing translational suppression, resulting in a small amount of full-length Snm1 protein. This small amount of Snm1 protein was sufficient to maintain enough RNase MRP activity to support viability. Translational suppression was accomplished in two ways. First, CEN plasmid missegregation leads to plasmid amplification, which in turn leads to SNM1 mRNA overexpression. Translational suppression of a small amount of the superabundant SNM1 mRNA results in sufficient Snm1 protein to support viability. CEN plasmid missegregation is believed to be the result of a prolonged telophase arrest that has been recently identified in RNase MRP mutants. Either the SNM1 gene is inherently susceptible to translational suppression or extremely small amounts of Snm1 protein are sufficient to maintain essential levels of MRP activity.     

Role of RNase MRP in viral RNA degradation and RNA recombination

RNA degradation, together with RNA synthesis, controls the steady-state level of viral RNAs in infected cells. The endoribonucleolytic cleavage of viral RNA is important not only for viral RNA degradation but for RNA recombination as well, due to the participation of some RNA degradation products in the RNA recombination process. To identify host endoribonucleases involved in degradation of Tomato bushy stunt virus (TBSV) in a Saccharomyces cerevisiae model host, we tested eight known endoribonucleases. Here we report that downregulation of SNM1, encoding a component of the RNase MRP, and a temperature-sensitive mutation in the NME1 gene, coding for the RNA component of RNase MRP, lead to reduced production of the endoribonucleolytically cleaved TBSV RNA in yeast. We also show that the highly purified yeast RNase MRP cleaves the TBSV RNA in vitro, resulting in TBSV RNA degradation products similar in size to those observed in yeast cells. Knocking down the NME1 homolog in Nicotiana benthamiana also led to decreased production of the cleaved TBSV RNA, suggesting that in plants, RNase MRP is involved in TBSV RNA degradation. Altogether, this work suggests a role for the host endoribonuclease RNase MRP in viral RNA degradation and recombination.     

RRP5 is required for formation of both 18S and 5.8S rRNA in yeast.

Three of the four eukaryotic ribosomal RNA molecules (18S, 5.8S and 25-28S) are synthesized as a single precursor which is subsequently processed into the mature rRNAs by a complex series of cleavage and modification reactions. In the yeast Saccharomyces cerevisiae, the early pre-rRNA cleavages at sites A0, A1 and A2, required for the synthesis of 18S rRNA, are inhibited in strains lacking RNA or protein components of the U3, U14, snR10 and snR30 small nucleolar ribonucleoproteins (snoRNPs). The subsequent cleavage at site A3, required for formation of the major, short form of 5.8S rRNA, is carried out by another ribonucleoprotein, RNase MRP. A screen for mutations showing synthetic lethality with deletion of the non-essential snoRNA, snR10, identified a novel gene, RRP5, which is essential for viability and encodes a 193 kDa nucleolar protein. Genetic depletion of Rrp5p inhibits the synthesis of 18S rRNA and, unexpectedly, also of the major short form of 5.8S rRNA. Pre-rRNA processing is concomitantly impaired at sites A0, A1, A2 and A3. This distinctive phenotype makes Rrp5p the first cellular component simultaneously required for the snoRNP-dependent cleavage at sites A0, A1 and A2 and the RNase MRP-dependent cleavage at A3 and provides evidence for a close interconnection between these processing events. Putative RRP5 homologues from Caenorhabditis elegans and humans were also identified, suggesting that the critical function of Rrp5p is evolutionarily conserved.     

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     

Characterization and purification of Saccharomyces cerevisiae RNase MRP reveals a new unique protein component

In the yeast Saccharomyces cerevisiae, RNase mitochondrial RNA processing (MRP) is an essential endoribonuclease that consists of one RNA component and at least nine protein components. Characterization of the complex is complicated by the fact that eight of the known protein components are shared with a related endoribonuclease, RNase P. To fully characterize the RNase MRP complex, we purified it to apparent homogeneity in a highly active state using tandem affinity purification. In addition to the nine known protein components, both Rpr2 and a protein encoded by the essential gene YLR145w were present in our preparations of RNase MRP. Precipitation of a tagged version of Ylr145w brought with it the RNase MRP RNA, but not the RNase P RNA. A temperature-sensitive ylr145w mutant was generated and found to exhibit a rRNA processing defect identical to that seen in other RNase MRP mutants, whereas no defect in tRNA processing was observed. Homologues of the Ylr145w protein were found in most yeasts, fungi, and Arabidopsis. Based on this evidence, we propose that YLR145w encodes a novel protein component of RNase MRP, but not RNase P. We recommend that this gene be designated RMP1, for RNase MRP protein 1.     

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.     

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.     

Lithium toxicity in yeast is due to the inhibition of RNA processing enzymes.

Hal2p is an enzyme that converts pAp (adenosine 3',5' bisphosphate), a product of sulfate assimilation, into 5' AMP and Pi. Overexpression of Hal2p confers lithium resistance in yeast, and its activity is inhibited by submillimolar amounts of Li+ in vitro. Here we report that pAp accumulation in HAL2 mutants inhibits the 5'-->3' exoribonucleases Xrn1p and Rat1p. Li+ treatment of a wild-type yeast strain also inhibits the exonucleases, as a result of pAp accumulation due to inhibition of Hal2p; 5' processing of the 5.8S rRNA and snoRNAs, degradation of pre-rRNA spacer fragments and mRNA turnover are inhibited. Lithium also inhibits the activity of RNase MRP by a mechanism which is not mediated by pAp. A mutation in the RNase MRP RNA confers Li+ hypersensitivity and is synthetically lethal with mutations in either HAL2 or XRN1. We propose that Li+ toxicity in yeast is due to synthetic lethality evoked between Xrn1p and RNase MRP. Similar mechanisms may contribute to the effects of Li+ on development and in human neurobiology.     

Processing of the yeast pre-rRNA at sites A(2) and A(3) is linked.

Cleavage of the yeast pre-rRNA at site A(2) in internal transcribed spacer 1 (ITS1) requires multiple snoRNP species, whereas cleavage at site A(3),located 72 nt 3' in ITS1, requires Rnase MRP. Analyses of mutations in the pre- rRNA have revealed an unexpected link between processing at A(2) and A(3). Small substitution mutations in the 3' flanking sequence at A(2) inhibit processing at site A(3), whereas a small deletion at A(3) has been shown to delay processing at site A(2). Moreover, the combination of mutations in cis at both A(2) and A(3) leads to the synthesis of pre-rRNA species with 5' ends within the mature 18S rRNA sequence, at sites between + 482 and + 496. The simultaneous interference with an snoRNP processing complex at site A(2) and an Rnase MPRP complex at site A(3) may activate a pre-rRNA breakdown pathway. The same aberantpre-rRNA species are observed in strains with mutations in the RNA component of Rnase MRP, consistent with interactions between the processing complexes. Furthermore, genetic depletion of the snoRNA, snR30, has been shown to affect the coupling between cleavage by Rnase MRP and subsequent exonuclease digestion.We conclude that an sno-RNP-dependent processing complex that is required for A(2) cleavage and that recognizes the 3' flanking sequence at A(2), interacts with the RNase MRP complex bound to the pre-rRNA around site A(3).     

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.     

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     

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.     

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.