The Yeast Nucleolar Protein Cbf5p Is Involved in rRNA Biosynthesis and Interacts Genetically with the RNA Polymerase I Transcription Factor RRN3

Abstract

Carbon, Mol. Cell. Biol. 13:4884-4893, 1993). Cbf5p also binds microtubules in vitro and interacts genetically with two known centromere-related protein genes (NDC10/CBF2 and MCK1). However, Cbf5p was found to be nucleolar and is highly homologous to the rat nucleolar protein NAP57, which coimmunoprecipitates with Nopp140 and which is postulated to be involved in nucleolarcytoplasmic shuttling (U. T. Meier, and G. Blobel, J. Cell Biol. 127:1505-1514, 1994). The temperaturesensitive cbf5-1 mutant demonstrates a pronounced defect in rRNA biosynthesis at restrictive temperatures, while tRNA transcription and pre-rRNA and pre-tRNA cleavage processing appear normal. The cbf5-1 mutant cells are deficient in cytoplasmic ribosomal subunits at both permissive and restrictive temperatures. A high-copy-number yeast genomic library was screened for genes that suppress the cbf5-1 temperature-sensitive growth phenotype. SYC1 (suppressor of yeast cbf5-1) was identified as a multicopy suppressor of cbf5-1 and subsequently was found to be identical to RRN3, an RNA polymerase I transcription factor. A cbf5⌬ null mutant is not rescued by plasmid pNOY103 containing a yeast 35S rRNA gene under the control of a Pol II promoter, indicating that Cbf5p has one or more essential functions in addition to its role in rRNA transcription.Cbf5p of the yeast Saccharomyces cerevisiae was originally isolated as one of the major low-affinity centromeric DNA (CEN) binding proteins (23). CBF5 is an essential gene encoding a highly charged protein with a domain containing ten tandem KKE/D repeats. These repeats are homologous to portions of microtubule-associated proteins 1A and 1B in the domain responsible for microtubule binding (40), and Cbf5p has been shown to bind microtubules in vitro (23). Yeast cells containing C-terminal truncated CBF5 genes delay, with replicated genomes, at the G 2 /M phase of the cell cycle, with the replicated DNA being located at the bud junction (23). Overexpression of Cbf5p suppresses the ndc10-1 temperature-sensitive (ts) mutation in the gene specifying the 110-kDa subunit (Cbf2p/Ndc10p) of the multisubunit yeast centromere DNA binding complex, CBF3, and overexpression of meiosis and centromere regulatory kinase Mck1p suppresses a ts mutation in the gene for either Cbf2p or Cbf5p (22). These genetic interactions support a direct or indirect link between Cbf5p and centromeres. However, both Cbf5p and a homologous protein (NAP57) from rats were found to be nucleolar proteins (21,33). While a functional relationship between a nucleolar protein and centromeres is not obvious, some proteins are associated with both the nucleolus and the centromere in higher eukaryotes. It has been known for some time that centromere autoantigens associate with the nucleolus (43), and experiments with autoimmune sera have also shown that a set of nucleolar proteins and ribonuclear proteins relocate around chromosomes during mitosis (16,17).The nucleolar location of Cbf5p may be indicative of a function unrelated to centromeres and chromos...

Cbf5p, a potential pseudouridine synthase, and Nhp2p, a putative RNA-binding protein, are present together with Gar1p in all H BOX/ACA-motif snoRNPs and constitute a common bipartite structure

“…Homologues of Gar1p share a central highly conserved domain but possess GAR domains of variable length+ Amino acid alignment of the S. cerevisiae (accession no+ X63617), S. pombe (accession no+ Z19576), human (accession no+ AA308727), Drosophila (accession no+ S49193), Cytosporidium (accession no+ AA532317), C. elegans (accession no+ C47427), and Arabidopsis (accession no+ N65654 and T21476) Gar1p homologues using the ClustalV program+ Identical residues are indicated in white on black boxes+ Conserved residues are indicated by grey boxes and are grouped as described in Schulz & Schirmer (1979)+ Conserved RGG tripeptides are indicated in white on dark grey boxes+ Amino acid positions are indicated on the left+ Dashed lines indicate spaces inserted to aid the alignment of the amino acid sequences+ Note that the human, Cytosporidium, and C. elegans sequences are incomplete at the N terminus and the Cytosporidium, C. elegans, and Arabidopsis sequences appear to be incomplete at the C terminus+ domain suggesting that these proteins may exhibit RNAbinding properties (Koonin et al+, 1994)+ The individual amino acids proposed to be involved in the interaction with RNA are indicated in Figure 6+ However, it should be noted that the amino acids in this motif are not the most conserved residues in this region of Nhp2p+ The presence of a putative RNA-binding domain in Nhp2p suggests that this protein may indeed directly bind the conserved H/ACA snoRNA sequences and function as the primary binding protein in snoRNP assembly+ The human sequence is considerably longer at the N terminus than the other eukaryotic counterparts; however, the N terminus of the Nhp2 protein homologues appears to be the least conserved region of the polypeptide+ Cbf5p is the most studied of the three H/ACA snoRNP core proteins+ It has been proposed to function in centromere binding (Jiang et al+, 1993); as an integral snoRNP protein (this work), ⌿ synthase (Koonin, 1996), or rRNA processing factor (Cadwell et al+, 1997); or in association with RNA polymerase I transcription factor RRN3 (Cadwell et al+, 1997) or with the nucleolar/ cytoplasmic shuttle protein Nopp140 (Meier & Blobel, 1994)+ Cbf5p homologues have been previously reported for S. cerevisae (Jiang et al+, 1993), Kluyveromyces lactis (Winkler et al+, 1998), and rat (NAP57) (Meier & Blobel, 1994)+ The database search revealed further homologues from a variety of eukaryotes and, surprisingly, archaebacteria (Fig+ 7A)+ In screening the Archaeal genome for ⌿ synthases homologous to Cbf5p, we noticed that TRUB also identifies the same Methanococcus ORF as the one presented in Figure 7A+ The yeast Cbf5p is 41% identical and 62% similar to the Methanococcus ORF while the yeast TRUB protein is only 16% identical and 29% similar to the Archaeal homologue+ Thus we conclude that the Archaeal protein is a Cbf5p homologue and not a TRUB homologue+ The finding of an Archaeal Cbf5p homologue raises the interesting possibility that Archaebacteria possess H/ACA snoRNP+ Alternatively, Cbf5p may be present in Archaea as a single protein not associated with RNP complexes+ Also included in the alignment in …”

“…Highly conserved homologues of Cbf5p share a high level of sequence similarity with known ⌿ synthases+ A: Amino acid alignment of the S. cerevisiae Cbf5p (accession no+ L12351), S. pombe Cbf5p (accession no+ Z97210), rat NAP57 (accession no+ Z34922), C. elegans (accession no+ Z92803), Drosophila (accession no+ AF017230), and Methanococcus (accession no+ U67472) Cbf5p/NAP57 protein homologues+ The sequences of the E. coli (accession no+ P09171) and Haemophilus influenza (accession no+ P45142) TRUB ⌿ synthases are also included as a comparison+ Identical residues are indicated in white on black boxes+ Conserved residues are indicated by grey boxes and are grouped as in Figure 5+ The boxed regions represent the two conserved regions found in the majority of ⌿ synthases+ Amino acid positions are indicated on the left+ Dashed lines indicate spaces inserted to align the amino acid sequences+ Only the homologous sequences have been included in this alignment+ B: Amino acid alignment of the highly charged C-terminal domains of Cbf5p homologues from S. cerevisiae, S. pombe, K. lactis (accession no+ AF008563), Dictyostelium discoidium (accession no+ C23692), rat, human (accession no+ U59151), Drosophila, and C. elegans+ KKE or KKD amino acid repeats are indicated in white on black boxes+ Other charged amino acids are indicated by grey boxes+ region of the snoRNA, and hence resemble the twodomain structure seen using the electron microscope (Fig+ 9)+ The extremely high degree of sequence identity seen between Cbf5p and other known ⌿ synthases suggests that this protein is indeed the enzyme activity responsible for snoRNA-directed modification (Koonin, 1996)+ While preparing this article, a paper describing Cbf5p as an integral H/ACA snoRNP protein involved in snoRNA biogenesis, 18S rRNA processing, and ⌿ synthesis was published (Lafontaine et al+, 1998)+ This suggests that the H/ACA snoRNPs are individual ⌿ synthases with the RNA component functioning in substrate recognition through direct base-pairing interactions+ The ⌿ synthases studied so far do not contain an RNA subunit and recognize the substrate through protein-RNA interactions (Kammen et al+, 1988;Nurse et al+, 1995;Wrzesinski et al+, 1995a,b;Simos et al+, 1996;Lecointe et al+, 1998)+ Hence, the H/ACA snoRNPs represent a novel type of modification enzyme+ In comparing the amino acid sequence of Cbf5p with known ⌿ synthases it is clear that the longer snoRNP protein contains essential highly conserved domains not present in the prokaryotic modification enzymes+ If one considers the fact that Cbf5p is essential for ribosome biogenesis and is proposed to interact with several other nucleolar proteins (Meier & Blobel, 1994;Cadwell et al+, 1997), this suggests that Cbf5p has other roles in the structure and function of this class of snoRNPs in addition to catalyzing ⌿ synthesis+ FIGURE 8. Electron microscopy of yeast snR30 and snR42 snoRNP complexes+ Purified snR30 and snR42 snoRNP complexes were negatively stained with uranyl formate+ Three common classes of (A) snR30 snoRNP and (B) snR42 snoRNP images+ For each row five examples of each class are shown of both the snR30 and snR42 complexes+ The corresponding rows in A and B show the respective particle in a similar projection form+ The left and mid...…”

“…The rRNA ⌿ synthase has yet to be identified; however, it is entirely possible that the protein responsible for the enzymatic activity is an integral component of the H/ACA snoRNPs+ The prokaryotic tRNA and rRNA ⌿ synthases do not use a guide RNA to recognize specific RNA substrates+ Multiple tRNA ⌿ synthases exist in prokaryotes that recognize either a single site or several sites with a similar structure (Kammen et al+, 1988;Nurse et al+, 1995;Wrzesinski et al+, 1995a,b;Simos et al+, 1996;Lecointe at al+, 1998)+ Therefore, the use of a guide RNA to direct rRNA ⌿ synthesis in eukaryotes is quite unique (Ganot et al+, 1997a;Ni et al+, 1997)+ It has recently been demonstrated that Gar1p, a protein common to all H/ACA snoRNPs, is required for ⌿ formation in yeast (Bousquet-Antonelli et al+, 1997)+ It is, however, highly unlikely that this protein is the modification enzyme, because Gar1p has little homology to known ⌿ synthases and is therefore postulated to function as an accessory protein in this event (Bousquet-Antonelli et al+, 1997)+ One possible candidate for the ⌿ synthase is Cbf5 (mammalian homologue NAP57; Meier & Blobel+, 1994), a nucleolar protein involved in ribosome biogenesis that shows a remarkably high degree of sequence similarity with known ⌿ synthases (Koonin, 1996;Cadwell et al+, 1997)+ Indeed, while we were preparing this article, a paper describing Cbf5p as an integral H/ACA snoRNP protein involved in snoRNA biogenesis, 18S rRNA processing, and ⌿ synthesis was published (Lafontaine et al+, 1998)…”

Actively transcribed rRNA genes in S. cerevisiae are organized in a specialized chromatin associated with the high-mobility group protein Hmo1 and are largely devoid of histone molecules

“…6A) and the isogenic RRN3 wildtype strain (CG379) (RRN3 in Fig. 6A; Cadwell et al 1997). The rrn3-ts strain supports rDNA transcription at 24°C, while incubation at 37°C leads to transcriptional shutdown, presumably because the initiation-competent Figure 6.…”

Rrp8p is a yeast nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2

“…Structure of the pre-rRNA and processing pathway in Saccharomyces cerevisiae. A: Structure of the 35S pre-rRNA+ The 35S operon contains the sequences for the mature 18S, 5+8S, and 25S rRNAs separated by the two internal transcribed spacers (ITS1 and ITS2)+ In addition, two external transcribed spacers, the 59ETS and 39ETS, are present at either end+ B: Pre-rRNA processing pathway+ The 35S pre-rRNA undergoes sequential processing reactions to generate the mature rRNAs+ Cleavage at A0 generates the 33S pre-rRNA+ It is subsequently cleaved at sites A1 at the 59 end of the 18S rRNA giving rise to the 32S pre-rRNA, and A2 in ITS1 yielding the 20S and 27SA2 pre-rRNAs+ The latter two species are precursors for the rRNAs respectively found in the small and large ribosomal subunits+ The 20S pre-rRNA is endonucleolytically cleaved at site D to produce the 18S rRNA+ The 27SA2 intermediate is processed by two alternative pathways+ In the major pathway, 27SA2 is cleaved by RNase MRP at site A3 in ITS1 yielding 27SA3, which is trimmed by 5939 exonucleases Rat1p and Xrn1p to site B1 S , generating the 27SB S precursor+ Approximately 15% of the 27SA2 molecules are processed at site B1 L , producing the 27SB L pre-rRNA+ 27SB S and 27SB L molecules are then processed at sites C1 and C2, yielding mature 25S rRNA and the 7S S and 7S L pre-rRNAs respectively, which are converted to 5+8S S and 5+8S L rRNAs by a complex of 39 59 exoribonucleases, the "exosome+" shifted to their associated proteins+ C/D snoRNPs are now known to contain three common essential proteins, Nop1p/fibrillarin (Tyc & Steitz, 1989, Baserga et al+, 1991Peculis & Steitz, 1994;Ganot et al+, 1997b), Nop56p, and Nop58p/Nop5p (Gautier et al+, 1997;Wu et al+, 1998, Lafontaine & Tollervey, 1999, whereas H/ACA snoRNPs contain at least four, Cbf5p (Jiang et al+, 1993;Cadwell et al+, 1997;, Gar1p (Girard et al+, 1992, Ganot et al+, 1997bHenras et al+, 1998), Nhp2p (Henras et al+, 1998;Watkins et al+, 1998), and Nop10p (Henras et al+, 1998)+ The discovery that Cbf5p, which is highly related to pseudouridine synthases (Koonin, 1996), is an integral snoRNP component strongly suggests that H/ACA snoRNPs not only select, but also modify prerRNA uridine residues )+ C/D snoRNPs also probably catalyze pre-rRNA modifications, as it has recently been noticed that the first characterized snoRNP protein component, Nop1p/ fibrillarin, contains the conserved methyltransferase motifs (Niewmierzycka & Clarke, 1999)+ SnoRNPs need to be able to reversibly interact with the pre-rRNA to catalyze pre-rRNA modifications or to promote pre-rRNA cleavage events+ Little is known about the ways snoRNPs/pre-rRNA interactions are modulated+ In the absence of the Gar1p protein, so-called because it contains two glycine/arginine-rich (GAR) domains at its N-and C-ter...…”