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...

Dot1 methylates histone H3 lysine 79 (H3K79) on the nucleosome core and is involved in Sir proteinmediated silencing. Previous studies suggested that H3K79 methylation within euchromatin prevents nonspecific binding of the Sir proteins, which in turn facilitates binding of the Sir proteins in unmethylated silent chromatin. However, the mechanism by which the Sir protein binding is influenced by this modification is unclear. We performed genome-wide synthetic genetic array (SGA) analysis and identified interactions of DOT1 with SIR1 and POL32. The synthetic growth defects found by SGA analysis were attributed to the loss of mating type identity caused by a synthetic silencing defect. By using epistasis analysis, DOT1, SIR1, and POL32 could be placed in different pathways of silencing. Dot1 shared its silencing phenotypes with the NatA N-terminal acetyltransferase complex and the conserved N-terminal bromo adjacent homology (BAH) domain of Sir3 (a substrate of NatA). We classified all of these as affecting a common silencing process, and we show that mutations in this process lead to nonspecific binding of Sir3 to chromatin. Our results suggest that the BAH domain of Sir3 binds to histone H3K79 and that acetylation of the BAH domain is required for the binding specificity of Sir3 for nucleosomes unmethylated at H3K79.Gene silencing in Saccharomyces cerevisiae at telomeres and the silent mating type loci is mediated by Sir proteins, which are recruited to DNA elements called silencers by sequencespecific DNA binding proteins (16,20,61). Upon the recruitment of Sir2 and Sir4 to silencers, Sir3 can bind, and the silent chromatin structure can subsequently spread in cis by interactions with neighboring nucleosomes (42). Silent chromatin in yeast is characterized by the absence of histone modifications, suggesting that the Sir complex preferentially binds to unmodified histones (16, 61). The NAD-dependent histone deacetylase activity of Sir2 is required for the spread and formation of a repressive Sir2-Sir3-Sir4 (Sir2/3/4) chromatin structure (35,42,74), and the binding of Sir3 to histone peptides in vitro has been shown to be negatively affected by the methylation and acetylation of the tails of histone H3 and H4 (8, 42, 63). Binding of Sir3 to histone tails is mediated by the C terminus of Sir3 (20). However, full-length Sir3 can bind to nucleosomes which lack histone tails, suggesting that Sir3 also interacts with other features of the nucleosome (23).In addition to modifications on the histone tails, silencing is positively affected by the methylation of lysine 79 of histone H3 (H3K79), a residue on the nucleosome core (15,39,47,49,76). The responsible methyltransferase Dot1 methylates ϳ90% of histone H3 and does so predominantly in euchromatin (39,47,49,76). In the absence of Dot1, binding of Sir2 and Sir3 at silent chromatin is reduced, and Sir3 becomes redistributed (47,49,62,76). We previously proposed that the methylated H3K79 (H3K79me) in euchromatin prevents nonspecific binding of Sir proteins to euchromatin...

Saccharomyces cerevisiae contains three nonessential genes (NGL1, NGL2, and NGL3 ) that encode proteins containing a domain with similarity to a Mg 21 -dependent endonuclease motif present in the mRNA deadenylase Ccr4p. We have investigated a possible role of these proteins in rRNA processing, because for many of the pre-rRNA processing steps, the identity of the responsible nuclease remains elusive. Analysis of RNA isolated from cells in which the NGL2 gene has been inactivated (ngl2D ) demonstrates that correct 39-end formation of 5.8S rRNA at site E is strictly dependent on Ngl2p. No role in pre-rRNA processing could be assigned to Ngl1p and Ngl3p. The 39-extended 5.8S rRNA formed in the ngl2D mutant is slightly shorter than the 6S precursor previously shown to accumulate upon combined deletion of the 39 r 59 exonuclease-encoding REX1 and REX2 genes or upon depletion of the exosomal subunits Rrp40p or Rrp45p. Thus, our data add a further component to the set of nucleases required for correct 39-end formation of yeast 5.8S rRNA.

No abstract

Rrp5p is the only protein so far known to be required for the processing of yeast pre-rRNA at both the early sites A0, A1 and A2 leading to 18S rRNA and at site A3, the first step specific for the pathway leading to 5.8S/25S rRNA. Previous in vivo mutational analysis of Rrp5p demonstrated that the first 8 of its 12 S1 RNA-binding motifs are involved in the formation of the 'short' form of 5.8S rRNA (5.8S(S)), which is the predominant species under normal conditions. We have constructed two strains in which the genomic RRP5 gene has been replaced by an rrp5 deletion mutant lacking either S1 motifs 3-5 (rrp5-Delta3) or 5-8 (rrp5-Delta4). The first mutant synthesizes almost exclusively 5.8S(L) rRNA, whereas the second one still produces a considerable amount of the 5.8S(S) species. Nevertheless, both mutations were found to block cleavage at site A3 completely. Instead, a novel processing event occurs at a site in a conserved stem-loop structure located between sites A2 and A3, which we have named A4. A synthetic lethality screen using the rrp5-Delta3 and rrp-Delta4 mutations identified the REX4 gene, which encodes a non-essential protein belonging to a class of related yeast proteins that includes several known 3'-->5' exonucleases. Inactivation of the REX4 gene in rrp5-Delta3 or rrp-Delta4 cells abolished cleavage at A4, restored cleavage at A3 and returned the 5.8S(S):5.8S(L) ratio to the wild-type value. The sl phenotype of the rrp5Delta/rex4(-) double mutants appears to be due to a severe disturbance in ribosomal subunit assembly, rather than pre-rRNA processing. The data provide direct evidence for a crucial role of the multiple S1 motifs of Rrp5p in ensuring the correct assembly and action of the processing complex responsible for cleavage at site A3. Furthermore, they clearly implicate Rex4p in both pre-rRNA processing and ribosome assembly, even though this protein is not essential for yeast.