Mutagenesis of SNM1, which encodes a protein component of the yeast RNase MRP, reveals a role for this ribonucleoprotein endoribonuclease in plasmid segregation
T. Cai et al., Mutagenesis of SNM1, which encodes a protein component of the yeast RNase MRP, reveals a role for this ribonucleoprotein endoribonuclease in plasmid segregation, MOL CELL B, 19(11), 1999, pp. 7857-7869
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.