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.
Cai
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Department of Biochemistry, State University of New York Health Science Center at Syracuse, Syracuse, New York 13210, USA.
Reilly
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Cerio
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Schmitt
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D015825:Chromosomes, Fungal / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004706:Endodeoxyribonucleases; D004722:Endoribonucleases / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D020043:Genes, Essential; D005800:Genes, Fungal; D016296:Mutagenesis; D009687:Nuclear Proteins; D010957:Plasmids / Q000235:genetics*; D011485:Protein Binding; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D012323:RNA Processing, Post-Transcriptional; D020871:RNA Stability; D012331:RNA, Fungal / Q000378:metabolism; D012340:RNA, Ribosomal, 5.8S / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D008941:Spindle Apparatus / Q000648:ultrastructure; D013489:Suppression, Genetic
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D009687:Nuclear Proteins; D012331:RNA, Fungal; D012340:RNA, Ribosomal, 5.8S; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins; D004706:Endodeoxyribonucleases; D004722:Endoribonucleases; C090162:PSO2 protein, S cerevisiae; C058330:mitochondrial RNA-processing endoribonuclease