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 ha ve roles in both mitochondrial DNA replication and nuclear 5.8S rRNA proces sing. SNM1 encodes an essential 22.5-kDa protein that is a component of yea st RNase MRP. It is an RNA binding protein that binds the MRP RNA specifica lly. This 198-amino-acid protein can be divided into three structural regio ns: a potential leucine zipper near the amino terminus, a binuclear zinc cl uster in the middle region, and a serine- and lysine-rich region near the c arboxy terminus. We have performed PCR mutagenesis of the SNM1 gene to prod uce 17 mutants that have a conditional phenotype for growth at different te mperatures. Yeast strains carrying any of these mutations as the only copy of snm1 display an rRNA processing defect identical to that in MRP RNA muta nts. 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 p romotes 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 requi red for processing of the 5.8S rRNA Surprisingly, a large number of conditi onal mutations that resulted from nonsense and frameshift mutations through out the coding regions were identified. The most severe of these was a fram eshift at amino acid 7. These mutations were found to be undergoing transla tional 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 accomplis hed in two ways. First, CEN plasmid missegregation leads to plasmid amplifi cation, which in turn leads to SNM1 mRNA overexpression. Translational supp ression of a small amount of the superabundant SNM1 mRNA results in suffici ent Snm1 protein to support viability. CEN plasmid missegregation is believ ed to be the result of a prolonged telophase arrest that has been recently identified in RNase MRP mutants. Either the SNM1 gene is inherently suscept ible to translational suppression or extremely small amounts of Snm1 protei n are sufficient to maintain essential levels of MRP activity.