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

Using molecular genetic techniques, we have generated and characterized six temperature sensitive (ts) alleles of nop2. All failed to support growth a t 37 degreesC 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 degreesC. Each allele contains bet ween seven and 14 amino acid substitutions and one possesses a nonsense mut ation near the C-terminus. Mapping experiments with one allele, nop2-4, rev ealed that a subset of the amino acid substitutions conferred the ts phenot ype and that these mutations have an additive effect. All six mutants exhib ited dramatic reductions in levels of 60S ribosome subunits under non-permi ssive conditions as well as some reduction at permissive temperature. Proce ssing of 27S pre-rRNA to mature 25S rRNA was defective in all six mutants g rown under nonpermissive conditions. Levels of the 40S ribosomal subunit an d 18S rRNA were not significantly affected. Amino acid substitutions in nop 2 conditional alleles are discussed in the context of the hypothesis that N op2p functions both as an RNA methyltransferase and a trans-acting factor i n rRNA processing and large ribosomal subunit biogenesis.