The final stage in the formation of the two large subunit rRNA species in Saccharomyces cerevisiae is the removal of internal transcribed spacer 2 (ITS2) from the 27SB precursors. This removal is initiated by endonucleolytic cleavage approximately midway in ITS2. The resulting 7S pre-rRNA, which is easily detectable, is then converted into 5.8S rRNA by the concerted action of a number of 3'-->5' exonucleases, many of which are part of the exosome. So far the complementary precursor to 25S rRNA resulting from the initial cleavage in ITS2 has not been detected and the manner of its conversion into the mature species is unknown. Using various yeast strains that carry different combinations of wild-type and mutant alleles of the major 5'-->3' exonucleases Rat1p and Xrn1p, we now demonstrate the existence of a short-lived 25.5S pre-rRNA whose 5' end is located closely downstream of the previously mapped 3' end of 7S pre-rRNA. The 25.5S pre-rRNA is converted into mature 25S rRNA by rapid exonucleolytic trimming, predominantly carried out by Rat1p. In the absence of Rat1p, however, the removal of the ITS2 sequences from 25.5S pre-rRNA can also be performed by Xrn1p, albeit somewhat less efficiently.

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The yeast nucleolar protein Rrp5p is the only known trans-acting factor that is essential for the synthesis of both 18S rRNA and the major, short form of 5.8S (5.8Ss) rRNA, which were thought to be produced in two independent sets of pre-rRNA processing reactions. To identify domains within Rrp5p required for either processing pathway, we have analyzed a set of eight deletion mutants that together cover the entire RRP5 sequence. Surprisingly, only one of the deletions is lethal, indicating that regions encompassing about 80% of the protein can be removed individually without disrupting its essential biological function. Biochemical analysis clearly demonstrated the presence of two distinct functional domains. Removal of each of three contiguous segments from the N-terminal half specifically inhibits the formation of 5.8Ss rRNA, whereas deleting part of the C-terminal region of the protein only blocks the production of 18S rRNA. The latter phenotype is also caused by a temperature-sensitive mutation within the same C-terminal region. The two functional regions identified by the mutational analysis appear to be correlated with the structural domains detected by computer analysis. They can even be physically separated, as demonstrated by the fact that full Rrp5p activity can be supplied by two contiguous protein fragments expressed in trans.

Saccharomyces cerevisiae Rio2p (encoded by open reading frame Ynl207w) is an essential protein of unknown function that displays significant sequence similarity to Rio1p/Rrp10p. The latter was recently shown to be an evolutionarily conserved, predominantly cytoplasmic serine/threonine kinase whose presence is required for the final cleavage at site D that converts 20 S pre-rRNA into mature 18 S rRNA. A data base search identified homologs of Rio2p in a wide variety of eukaryotes and Archaea. Detailed sequence comparison and in vitro kinase assays using recombinant protein demonstrated that Rio2p defines a subfamily of protein kinases related to, but both structurally and functionally distinct from, the one defined by Rio1p. Failure to deplete Rio2p in cells containing a GAL-rio2 gene and direct analysis of Rio2p levels by Western blotting indicated the protein to be low abundant. Using a GAL-rio2 gene carrying a point mutation that reduces the kinase activity, we found that depletion of this mutant protein blocked production of 18 S rRNA due to inhibition of the cleavage of cytoplasmic 20 S pre-rRNA at site D. Production of the large subunit rRNAs was not affected. Thus, Rio2p is the second protein kinase that is essential for cleavage at site D and the first in which the processing defect can be linked to its enzymatic activity. Contrary to Rio1p/Rrp10p, however, Rio2p appears to be localized predominantly in the nucleus.Like their counterparts in other eukaryotes, Saccharomyces cerevisiae ribosomes contain four species of rRNA: 5 S, 5.8 S, 18 S, and 25 S rRNAs. The genes encoding these rRNAs are organized on the yeast genome in 150 -200 tandem repeats, each of which comprises two transcriptional units separated by non-transcribed spacers. One of these units consists of a 5 S rRNA gene, transcribed by RNA polymerase III. The other unit contains single genes for each of the mature 18 S, 5.8 S, and 25 S rRNAs that are separated by internal transcribed spacers 1 and 2, whereas external transcribed spacer regions are present at either end of the unit (see Fig. 1A). After transcription of this polycistronic unit by RNA polymerase I, the spacers are removed from the primary transcript via an ordered series of endo-and exonucleolytic cleavages (see Fig. 1B) (reviewed in Refs. 1 and 2). The first detectable precursor species is 35 S pre-rRNA, which results from a cleavage at site B 0 in the 3Ј-external transcribed spacer by the yeast RNase III homolog Rnt1p (3,4). Subsequent cleavage at sites A 0 and A 1 in the 5Ј-external transcribed spacer first gives rise to 33 S and then 32 S pre-rRNA. The latter is cleaved at site A 2 to produce separate 20 S and 27 S A 2 precursors for the small and large ribosomal subunit, respectively.The majority (90%) of the 27 S A 2 precursor molecules are cleaved endonucleolytically at site A 3 , followed by exonucleolytic trimming to B 1S . The remainder are processed endonucleolytically 1 at site B 1L . The resulting 27 S B S and 27 S B L precursors, whose 5Ј-ends are located 6 nucleotide...