The 1:1 balance between the numbers of large and small ribosomal subunits can be disturbed by mutations that inhibit the assembly of only one of the subunits. Here, we have investigated if the cell can counteract an imbalance of the number of the two subunits. We show that abrogating 60S assembly blocks 40S subunit accumulation. In contrast, cessation of the 40S pathways does not prevent 60S accumulation, but does, however, lead to fragmentation of the 25S rRNA in 60S subunits and formation of a 55S ribosomal particle derived from the 60S. We also present evidence suggesting that these events occur post assembly and discuss the possibility that the turnover of subunits is due to vulnerability of free subunits not paired with the other subunit to form 80S ribosomes.

RNase MRP is a nucleolar RNA–protein enzyme that participates in the processing of rRNA during ribosome biogenesis. Previous experiments suggested that RNase MRP makes a nonessential cleavage in the first internal transcribed spacer. Here we report experiments with new temperature-sensitive RNase MRP mutants in Saccharomyces cerevisiae that show that the abundance of all early intermediates in the processing pathway is severely reduced upon inactivation of RNase MRP. Transcription of rRNA continues unabated as determined by RNA polymerase run-on transcription, but the precursor rRNA transcript does not accumulate, and appears to be unstable. Taken together, these observations suggest that inactivation of RNase MRP blocks cleavage at sites A0, A1, A2, and A3, which in turn, prevents precursor rRNA from entering the canonical processing pathway (35S > 20S + 27S > 18S + 25S + 5.8S rRNA). Nevertheless, at least some cleavage at the processing site in the second internal transcribed spacer takes place to form an unusual 24S intermediate, suggesting that cleavage at C2 is not blocked. Furthermore, the long form of 5.8S rRNA is made in the absence of RNase MRP activity, but only in the presence of Xrn1p (exonuclease 1), an enzyme not required for the canonical pathway. We conclude that RNase MRP is a key enzyme for initiating the canonical processing of precursor rRNA transcripts, but alternative pathway(s) might provide a backup for production of small amounts of rRNA.

Nearly half of ribosomal proteins are composed of a domain on the ribosome surface and a loop or extension that penetrates into the organelle's RNA core. Our previous work showed that ribosomes lacking the loops of ribosomal proteins uL4 or uL22 are still capable of entering polysomes. However, in those experiments we could not address the formation of mutant ribosomes, because we used strains that also expressed wild-type uL4 and uL22. Here, we have focused on ribosome assembly and function in strains in which loop deletion mutant genes are the only sources of uL4 or uL22 protein. The uL4 and uL22 loop deletions have different effects, but both mutations result in accumulation of immature particles that do not accumulate in detectable amounts in wild-type strains. Thus, our results suggest that deleting the loops creates kinetic barriers in the normal assembly pathway, possibly resulting in assembly via alternate pathway(s). Furthermore, deletion of the uL4 loop results in cold-sensitive ribosome assembly and function. Finally, ribosomes carrying either of the loop-deleted proteins responded normally to the secM translation pausing peptide, but the uL4 mutant responded very inefficiently to the cmlA^crb pause peptide.