Temperature sensitive nop2 alleles defective in synthesis of 25S rRNA and large ribosomal subunits in Saccharomyces cerevisiae

Abstract

Using molecular genetic techniques, we have generated and characterized six temperature sensitive (ts) alleles of nop2. All failed to support growth at 37°C and one was also formamide sensitive (fs) and failed to grow on media containing 3% formamide. Conditional lethality is not due to rapid turnover of mutant Nop2p proteins at 37°C. Each allele contains between seven and 14 amino acid substitutions and one possesses a nonsense mutation near the C-terminus. Mapping experiments with one allele, nop2-4, revealed that a subset of the amino acid substitutions conferred the ts phenotype and that these mutations have an additive effect. All six mutants exhibited dramatic reductions in levels of 60S ribosome subunits under non-permissive conditions as well as some reduction at permissive temperature. Processing of 27S pre-rRNA to mature 25S rRNA was defective in all six mutants grown under non-permissive conditions. Levels of the 40S ribosomal subunit and 18S rRNA were not significantly affected. Amino acid substitutions in nop2 conditional alleles are discussed in the context of the hypothesis that Nop2p functions both as an RNA methyltransferase and a trans-acting factor in rRNA processing and large ribosomal subunit biogenesis.