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

Abstract

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.

Characterization and Purification of Saccharomyces cerevisiae RNase MRP Reveals a New Unique Protein Component

“…8, a defect in 5.8S rRNA processing was observed in the rmp1-6 mutant at both the permissive and non-permissive temperatures. This change in the ratio of 5.8S rRNA species correlates well with the phenotype seen in other RNase MRP mutants (2,11,20,31). These results demonstrate that Rmp1p is required for the function of the RNase MRP enzyme in rRNA processing.…”

“…Strains and Media-Yeast media and genetic manipulations have been described previously (10,20). The Escherichia coli strain used for cloning, DH5␣, has the genotype 80dlacZ⌬M15 endA1 recA1 hsdR17 (r k Ϫ m k ϩ ) supE44 thi-1 Ϫ gyrA96 relA1 ⌬(lacIZYA-argF)U169 F Ϫ .…”

“…To purify RNase MRP, the YSW1 strain, which has the genotype MATa POP4::TAPTAG::TRP1ks pep4:LEU2 nuc1::LEU2 sep1::URA3 trp1his3 -11,15 can-100 ura3-1 leu2-3,112, was used (10). This strain was constructed by using PCR to amplify the TAP fusion cassette from pBS1479 (a gift from Bertrand Séraphin) and then using PCR-based genomic TAP tagging to integrate the tag into the carboxyl-terminal codon of the POP4 gene (19 Whole-cell Western Blot Analysis-Whole-cell yeast protein extracts were prepared as described previously (20). Samples were denatured in 2% (w/v) SDS and 5% (v/v) 2-mercaptoethanol for 5 min at 95°C and resolved on a 12% polyacrylamide gel (21).…”

“…Mutants that exhibited a temperature-conditional growth phenotype were selected for further analysis. The YCplac111 plasmid was recovered from the mutant yeast strain via transformation of E. coli with yeast cell lysates (20). The isolated plasmid was re-transformed into KLS111 to ensure that the mutations were linked to the plasmid.…”

Human ribonuclease P: Subunits, function, and intranuclear localization

“…The nucleolus is a storage site for complexes required for cell mitosis (Bachant & Elledge, 1999) and Cajal bodies are implicated in gene transcription in a cellcycle-dependent manner + These functions may also be relevant for some RNase P subunits concentrated in nucleoli and Cajal bodies+ The fact that the nucleolus disassembles before the onset of mitosis, thus forming perichromosomal regions and nucleolus-derived foci (Dundr et al+, 2000), and that hPop1 is associated with chromatids and the mitotic spindle region during anaphase (Dundr & Olson, 1998), raises the question if RNase P, RNase MRP, or their individual subunits have roles in the cell cycle+ Such a functional connection is supported by the recent finding that mutations in the RNA subunit of human RNase MRP cause a pleiotropic genetic disorder, cartilage hair hypoplasia, which is manifested by abnormal body development, defective immunity, and predisposition to several types of cancer (Ridanpaa et al+, 2001)+ In addition, genetic studies in S. cerevisiae reveal that Snm1, a protein subunit of RNase MRP, has a role in plasmid segregation and control of cell division (Cai et al+, 1999;Clayton, 2001)+ New functions are attributed to Rpm2, the protein subunit of the S. cerevisiae mitochondrial RNase P+ Genetic studies relate Rpm2p to mitochondrial protein synthesis and protein degradation through the proteasome function (Lutz et al+, 2000;Stribinskis et al+, 2001)+…”