RNA12-1 is a dominant temperature-sensitive (Ts) yeast mutant which has previously been reported to exhibit a defect in RNA accumulation at 37 degrees C. We further characterized this mutant through analyses of rRNA transcription rates and maturation. The results show that pre-rRNA is normally synthesized but that subsequent maturation is severely affected by a temperature upshift: the nascent rRNAs are under-methylated and little mature rRNA can be observed at 37 degrees C. Likewise, the accumulation of some mRNAs for ribosomal proteins is also prevented at 37 degrees C. The RNA12-1 mutation is recessive at 32 degrees C, which made it possible to clone the wild-type rna12+ gene by complementation of the Ts phenotype with plasmids from a multicopy yeast genomic library. The predicted gene product is a protein of 96,630 Da with no significant sequence similarity to any known proteins. Gene disruption is not lethal at either the permissive or the restrictive temperature. The gene is located on chromosome XIII, downstream of the ADH2 gene and 10 cM from the ADE4 gene. Furthermore, the mutant allele RNA12-1 was cloned and sequenced. A point mutation found in this allele leads to dominant thermosensitivity at 37 degrees C when the mutant gene is introduced into a wild-type strain. Taken together, these data suggest that the rna12+ gene product plays a dispensable role in early maturation of pre-rRNA but that its mutant gene product can interfere with the normal function of other proteins required for pre-rRNA maturation.