Structural and thermodynamic evidence for a stabilizing role of Nop5p in S-adenosyl-L-methionine binding to fibrillarin

In Archaea, fibrillarin and Nop5p form the core complex of box C/D small ribonucleoprotein particles, which are responsible for site-specific 2'-hydroxyl methylation of ribosomal and transfer RNAs. Fibrillarin has a conserved methyltransferase fold and employs S-adenosyl-l-methionine (AdoMet) as the cofactor in methyl transfer reactions. Comparison between recently determined crystal structures of free fibrillarin and fibrillarin-Nop5p-AdoMet tertiary complex revealed large conformational differences at the cofactor-binding site in fibrillarin. To identify the structural elements responsible for these large conformational differences, we refined a crystal structure of Archaeoglobus fulgidus fibrillarin-Nop5p binary complex at 3.5 A. This structure exhibited a pre-formed backbone geometry at the cofactor binding site similar to that when the cofactor is bound, suggesting that binding of Nop5p alone to fibrillarin is sufficient to stabilize the AdoMet-binding pocket. Calorimetry studies of cofactor binding to fibrillarin alone and to fibrillarin-Nop5p binary complex provided further support for this role of Nop5p. Mutagenesis and thermodynamic data showed that a cation-pi bridge formed between Tyr-89 of fibrillarin and Arg-169 of Nop5p, although dispensable for in vitro methylation activity, could partially account for the enhanced binding of cofactor to fibrillarin by Nop5p. Finally, assessment of cofactor-binding thermodynamics and catalytic activities of enzyme mutants identified three additional fibrillarin residues (Thr-70, Glu-88, and Asp-133) to be important for cofactor binding and for catalysis.     

The GTP-binding protein YqeH participates in biogenesis of the 30S ribosome subunit in Bacillus subtilis

Bacterial genome sequencing has revealed a novel family of P-loop GTPases that are often essential for growth. Accumulating evidence suggests that these proteins are involved in biogenesis of the 30S or 50S ribosomal subunits. YqeH is a member of this Obg/Era GTPase family, with its function remains to be uncovered. Here, we present results showing that YqeH is involved in the 30S subunit biogenesis in Bacillus subtilis. We observed a reduction in the 70S ribosome and accumulation of the free 50S subunit in YqeH-depleted cells. Interestingly, no free 30S subunit accumulation was evident. Consistent with the theory that YqeH is involved in 30S subunit biogenesis, a precursor of 16S rRNA and its degradation products were detected. Additionally, the reduction of free 30S subunit was not observed in Era-depleted cells. YqeH overexpression did not compensate for growth defects in mutants devoid of Era and vice versa. Moreover, in vitro GTPase analyses showed that YqeH possessed high intrinsic GTPase activity. In contrast, Era showed slow GTPase activity, which was enhanced by the 30S ribosomal subunit. Our findings strongly suggest that YqeH and Era function at distinct checkpoints during 30S subunit assembly. B. subtilis yqeH is classified as an essential gene due to the inability of the IPTG-dependent P(spac)-yqeH mutant to grow on LB or PAB agar plates in the absence of IPTG. However, in our experiments, the P(spac)-yqeH mutant grew in PAB liquid medium without IPTG supplementation, albeit at an impaired rate. This finding raises the interesting possibility that YqeH participates in assembly of the 30S ribosomal subunit as well as other cellular functions essential for growth on solid media.     

Nucleolar protein Nop12p participates in synthesis of 25S rRNA in Saccharomyces cerevisiae

A genetic screen for mutations synthetically lethal with temperature sensitive alleles of nop2 led to the identification of the nucleolar proteins Nop12p and Nop13p in Saccharomyces cerevisiae. NOP12 was identified by complementation of a synthetic lethal growth phenotype in strain YKW35, which contains a single nonsense mutation at codon 359 in an allele termed nop12-1. Database mining revealed that Nop12p was similar to a related protein, Nop13p. Nop12p and Nop13p are not essential for growth and each contains a single canonical RNA recognition motif (RRM). Both share sequence similarity with Nsr1p, a previously identified, non-essential, RRM-containing nucleolar protein. Likely orthologs of Nop12p were identified in Drosophila and Schizosaccharomyces pombe. Deletion of NOP12 resulted in a cold sensitive (cs) growth phenotype at 15°C and slow growth at 20 and 25°C. Growth of a nop12Δ strain at 15 and 20°C resulted in impaired synthesis of 25S rRNA, but not 18S rRNA. A nop13 null strain did not produce an observable growth phenotype under the laboratory conditions examined. Epitope-tagged Nop12p, which complements the cs growth phenotype and restores normal 25S rRNA levels, was localized to the nucleolus by immunofluorescence microscopy. Epitope-tagged Nop13p was distributed primarily in the nucleolus, with a lesser portion localizing to the nucleoplasm. Thus, Nop12p is a novel nucleolar protein required for pre-25S rRNA processing and normal rates of cell growth at low temperatures.     

Identification of nucleolus-localized PTEN and its function in regulating ribosome biogenesis

The tumor suppressor PTEN is a lipid phosphatase that is found mutated in different types of human cancers. PTEN suppresses cell proliferation by inhibiting the PI3K-Akt signaling pathway at the cell membrane. However, PTEN is also demonstrated to localize in the cell nucleus where it exhibits tumor suppressive activity via a different, unknown mechanism. In this study we report that PTEN also localizes to the nucleolus and that nucleolar PTEN plays an important role in regulating nucleolar homeostasis and maintaining nucleolar morphology. Overexpression of nuclear PTEN in PTEN null cells inhibits Akt phosphorylation and reduces cell size. Knockdown of PTEN in PTEN positive cells leads to nucleolar morphologic changes and an increase in the proportion of cells with a greater number of nucleoli. In addition, knockdown of PTEN in PTEN positive cells increased ribosome biogenesis. These findings expand current understanding of function and relevance of nuclear localized PTEN and provide a foundation for the development of novel therapies targeting PTEN.     

Human fibrillarin forms a sub-complex with splicing factor 2-associated p32, protein arginine methyltransferases, and tubulins alpha 3 and beta 1 that is independent of its association with preribosomal ribonucleoprotein complexes

Fibrillarin (FIB, Nop1p in yeast) is an RNA methyltransferase found not only in the fibrillar region of the nucleolus but also in Cajal bodies. FIB is essential for efficient processing of preribosomal RNA during ribosome biogenesis, although its precise function in this process and its role in Cajal bodies remain uncertain. Here, we demonstrate that the human FIB N-terminal glycine- and arginine-rich domain (residues 1-77) and its spacer region 1 (78-132) interact with splicing factor 2-associated p32 (SF2A-p32) and that the FIB methyltransferase-like domain (133-321) interacts with protein-arginine methyltransferase 5 (PRMT5, Janus kinase-binding protein 1). We also show that these proteins associate with several additional proteins, including PRMT1, tubulin alpha 3, and tubulin beta 1 to form a sub-complex that is principally independent of the association of FIB with preribosomal ribonucleoprotein complexes that co-immunoprecipitate with the sub-complex in human cells expressing FLAG-tagged FIB. Based on the physical association of FIB with SF2A-p32 and PRMTs, as well as the other reported results, we propose that FIB may coordinate both RNA and protein methylation during the processes of ribosome biogenesis in the nucleolus and RNA editing such as small nuclear (nucleolar) ribonucleoprotein biogenesis in Cajal bodies.     

Nop2p is required for pre-rRNA processing and 60S ribosome subunit synthesis in yeast.

To investigate the function of the nucleolar protein Nop2p in Saccharomyces cerevisiae, we constructed a strain in which NOP2 is under the control of a repressible promoter. Repression of NOP2 expression lengthens the doubling time of this strain about fivefold and reduces steady-state levels of 60S ribosomal subunits, 80S ribosomes, and polysomes. Levels of 40S subunits increase as the free pool of 60S subunits is reduced. Nop2p depletion impairs processing of the 35S pre-rRNA and inhibits processing of 27S pre-rRNA, which results in lower steady-state levels of 25S rRNA and 5.8S rRNA. Processing of 20S pre-rRNA to 18S rRNA is not significantly affected. Processing at sites A2, A3, B1L, and B1S and the generation of 5' termini of different pre-rRNA intermediates appear to be normal after Nop2p depletion. Sequence comparisons suggest that Nop2p may function as a methyltransferase. 2'-O-ribose methylation of the conserved site UmGm psi UC2922 is known to take place during processing of 27S pre-rRNA. Although Nop2p depletion lengthens the half-life of 27S pre-RNA, methylation of UmGm psi UC2922 in 27S pre-rRNA is low during Nop2p depletion. However, methylation of UmGm psi UC2922 in mature 25S rRNA appears normal. These findings provide evidence for a close interconnection between methylation at this conserved site and the processing step that yields the 25S rRNA.     

Spb1p is a yeast nucleolar protein associated with Nop1p and Nop58p that is able to bind S-adenosyl-L-methionine in vitro

We present here the characterization of SPB1, an essential yeast gene that is required for ribosome synthesis. A cold-sensitive allele for that gene (referred to here as spb1-1) had been previously isolated as a suppressor of a mutation affecting the poly(A)-binding protein gene (PAB1) and a thermosensitive allele (referred to here as spb1-2) was isolated in a search for essential genes required for gene silencing in Saccharomyces cerevisiae. The two mutants are able to suppress the deletion of PAB1, and they both present a strong reduction in their 60S ribosomal subunit content. In an spb1-2 strain grown at the restrictive temperature, processing of the 27S pre-rRNA into mature 25S rRNA and 5.8S is completely abolished and production of mature 18S is reduced, while the abnormal 23S species is accumulated. Spb1p is a 96.5-kDa protein that is localized to the nucleolus. Coimmunoprecipitation experiments show that Spb1p is associated in vivo with the nucleolar proteins Nop1p and Nop5/58p. Protein sequence analysis reveals that Spb1p possesses a putative S-adenosyl-L-methionine (AdoMet)-binding domain, which is common to the AdoMet-dependent methyltransferases. We show here that Spb1p is able to bind [(3)H]AdoMet in vitro, suggesting that it is a novel methylase, whose possible substrates will be discussed.     

Yeast Rrp14p is a nucleolar protein involved in both ribosome biogenesis and cell polarity

We previously cloned RRP14/YKL082c, whose product exhibits two-hybrid interaction with Ebp2p, a regulatory factor of assembly of 60S ribosomal subunits. Depletion of Rrp14p results in shortage of 60S ribosomal subunits and retardation of processing from 27S pre-rRNA to 25S rRNA. Furthermore, 35S pre-rRNA synthesis appears to decline in Rrp14p-depleted cells. Rrp14p interacts with regulatory factors of 60S subunit assembly and also with Utp11p and Faf1p, which are regulatory factors required for assembly of 40S ribosomal subunits. We propose that Rrp14p is involved in ribosome synthesis from the beginning of 35S pre-rRNA synthesis to assembly of the 60S ribosomal subunit. Disruption of RRP14 causes an extremely slow growth rate of the cell, a severe defect in ribosome synthesis, and a depolarized localization of cortical actin patches throughout the cell cycle. These results suggest that Rrp14p has dual functions in ribosome synthesis and polarized cell growth.     

Eukaryotic initiation factor 6, an evolutionarily conserved regulator of ribosome biogenesis and protein translation

We recently identified Receptor for Activated C Kinase 1 (RACK1) as one of the molecular links between abscisic acid (ABA) signaling and its regulation on protein translation. Moreover, we identified Eukaryotic Initiation Factor 6 (eIF6) as an interacting partner of RACK1. Because the interaction between RACK1 and eIF6 in mammalian cells is known to regulate the ribosome assembly step of protein translation initiation, it was hypothesized that the same process of protein translation in Arabidopsis is also regulated by RACK1 and eIF6. In this article, we analyzed the amino acid sequences of eIF6 in different species from different lineages and discovered some intriguing differences in protein phosphorylation sites that may contribute to its action in ribosome assembly and biogenesis. In addition, we discovered that, distinct from non-plant organisms in which eIF6 is encoded by a single gene, all sequenced plant genomes contain two or more copies of eIF6 genes. While one copy of plant eIF6 is expressed ubiquitously and might possess the conserved function in ribosome biogenesis and protein translation, the other copy seems to be only expressed in specific organs and therefore may have gained some new functions. We proposed some important studies that may help us better understand the function of eIF6 in plants.Key words: CK1, eIF6, PKC, protein translation, RACK1, ribosome assembly, ribosome biogenesisEukaryotic Initiation Factor 6 (eIF6) was originally purified from wheat germ¹ and was found to function as a ribosome dissociation factor through binding to the 60S ribosome subunit and preventing its association with the 40S ribosome subunit.² Its homologous proteins were later purified from rabbit reticulocyte lysates,³ calf liver⁴ and human cells.⁴ The action of eIF6 in preventing the ribosome subunits association was later found to involve another two proteins, the activated Protein Kinase C (PKC) and the Receptor for Activated C Kinase 1 (RACK1) in mammalian cells.⁵ PKC is a family of proteins that can be activated by elevated cellular concentration of Ca²⁺ or diacylglycerol and is involved in multiple signal transduction pathways in mammalian cells.⁶ RACK1 was identified as a receptor for activated PKC, anchoring PKC to the subcellular location where its substrate is present.^7,8 In this protein complex, RACK1 serves as a scaffold protein that simultaneously binds to eIF6 and activated PKC to bring these two proteins to close proximity. PKC then phosphorylates eIF6, leading to its dissociation from the 60S ribosome subunit and consequently allowing the association between the 40S and 60S ribosome subunits to assemble a functional 80S subunit to initiate protein translation⁵ (Fig. 1). Genetic studies supported the role of mammalian eIF6 in protein translation initiation as well as in cell growth.⁹ More recent structural studies supported a similar role of eIF6 in regulating 80S ribosome assembly in yeast.^10,11 Yeast eIF6 (Tif6p) was also known to regulate 60S ribosome biogenesis.^12,13 Very recently, eIF6 was identified as a component of a protein complex that interacts with the RNA-induced silencing complex and plays a role in microRNA-directed gene silencing.¹⁴ For a more comprehensive review of eIF6''s function in mammalian cells and in yeast, readers should refer to the following review article.¹⁵Open in a separate windowFigure 1A schematic presentation of the proposed molecular mode of action of eIF6, RACK1, PKC and CK1 in ribosome assembly and protein translation. Nuclear CK1 phosphorylates eIF6 at Serine 174 and Serine 175. This phosphorylation is required for the shuttling of eIF6 from nucleus into cytosol. eIF6 prevents joining of the cytosolic 60S ribosome subunit with the 40S subunit from forming a functional 80S ribosome. RACK1, via binding simultaneously to the eIF6 and the activated PKC, can facilitate the phosphorylation of eIF6 at Serine 235 by PKC. The Serine 235 phosphorylated eIF6 then disassociates from 60S ribosome, thus allowing the assembly of functional 80S ribosome and initiation of protein translation. CK1, Casein Kinase 1; 60S, 60S ribosome subunit; 40S, 40S ribosome subunit; eIF6, Eukaryotic Initiation Factor 6; RACK1, Receptor for Activated C Kinase 1; PKC, Protein Kinase C; pi, phosphate.Despite considerable progress that has been made in the identification of central components of plant hormone abscisic acid (ABA) signaling, little is known about the molecular mechanism of the long-recognized effect of ABA on protein translation. Our group has been working on the functional analysis of Arabidopsis RACK1 gene family,^16–19 and has identified RACK1 as a negative regulator of ABA responses.¹⁸ Recently, we discovered that RACK1 may play a role in ribosome assembly and 60S ribosome subunit biogenesis and therefore serve as one of the molecular links between ABA signaling and its control on protein translation.²⁰ In addition, we discovered that RACK1 physically interacts with eIF6 in a yeast two-hybrid assay and in a Bi-molecular Fluorescence Complementation assay in an Arabidopsis leaf mesophyll protoplast system. The conserved interaction between RACK1 and eIF6 in plants and in mammals implies an evolutionarily conserved role of eIF6 and RACK1 in ribosome biogenesis, assembly and protein translation.     

Cyclophilin acts as a ribosome biogenesis factor by chaperoning the ribosomal protein (PlRPS15) in filamentous fungi

The rapid transport of ribosomal proteins (RPs) into the nucleus and their efficient assembly into pre-ribosomal particles are prerequisites for ribosome biogenesis. Proteins that act as dedicated chaperones for RPs to maintain their stability and facilitate their assembly have not been identified in filamentous fungi. PlCYP5 is a nuclear cyclophilin in the nematophagous fungus Purpureocillium lilacinum, whose expression is up-regulated during abiotic stress and nematode egg-parasitism. Here, we found that PlCYP5 co-translationally interacted with the unassembled small ribosomal subunit protein, PlRPS15 (uS19). PlRPS15 contained an eukaryote-specific N-terminal extension that mediated the interaction with PlCYP5. PlCYP5 increased the solubility of PlRPS15 independent of its catalytic peptide-prolyl isomerase function and supported the integration of PlRPS15 into pre-ribosomes. Consistently, the phenotypes of the PlCYP5 loss-of-function mutant were similar to those of the PlRPS15 knockdown mutant (e.g. growth and ribosome biogenesis defects). PlCYP5 homologs in Arabidopsis thaliana, Homo sapiens, Schizosaccharomyces pombe, Sclerotinia sclerotiorum, Botrytis cinerea and Metarhizium anisopliae were identified. Notably, PlCYP5-PlRPS15 homologs from three filamentous fungi interacted with each other but not those from other species. In summary, our data disclosed a unique dedicated chaperone system for RPs by cyclophilin in filamentous fungi.     

Mitosis-specific phosphorylation of p60c-src by p34cdc2-associated protein kinase

As cells enter mitosis, the protein-tyrosine kinase, p60c-src, is known to be extensively phosphorylated on threonine in its amino-terminal region. In the present work, extracts of mitotic cells were searched for the protein kinase responsible for this phosphorylation. HeLa cells and Xenopus eggs were found to contain a mitosis-specific protein kinase activity capable of phosphorylating highly purified p60c-src in vitro on threonine residues. Tryptic phosphopeptide maps indicate that the mitotic HeLa kinase phosphorylates the same sites in vitro as those used during mitosis in vivo. In addition, this mitotic HeLa kinase comigrates on gel filtration with p34cdc2-associated histone H1 kinase, a well known regulator of mitotic events. Finally, antibodies to the C-terminal peptide of human p34cdc2 specifically deplete p60c-src-phosphorylating activity from mitotic extracts. These results suggest that p60c-src may act as an effector of p34cdc2 in certain mitotic processes.     

Kes1p shares homology with human oxysterol binding protein and participates in a novel regulatory pathway for yeast Golgi-derived transport vesicle biogenesis.

The yeast phosphatidylinositol transfer protein (Sec14p) is required for biogenesis of Golgi-derived transport vesicles and cell viability, and this essential Sec14p requirement is abrogated by inactivation of the CDP-choline pathway for phosphatidylcholine biosynthesis. These findings indicate that Sec14p functions to alleviate a CDP-choline pathway-mediated toxicity to yeast Golgi secretory function. We now report that this toxicity is manifested through the action of yeast Kes1p, a polypeptide that shares homology with the ligand-binding domain of human oxysterol binding protein (OSBP). Identification of Kes1p as a negative effector for Golgi function provides the first direct insight into the biological role of any member of the OSBP family, and describes a novel pathway for the regulation of Golgi-derived transport vesicle biogenesis.     

Separation of protein synthesis initiation factor eIF4A from a p220-associated cap binding complex activity

A cap binding complex activity was purified from HeLa cells by a procedure which does not depend on the use of cap-affinity chromatography. The activity co-purified with a Mr 220000 polypeptide (p220), but not with elF4A. The active complex therefore differs from eIF4F, the complex purified by cap analog-affinity chromatography, in that it lacks the Mr 50000 subunit which is antigenically identical to elF4A. The activities of elF4F, CBP I and the eIF4A free complex purified here were compared in a fractionated system translating capped globin mRNA. Results indicate that the two complexes have similar activities and that they perform a function which cannot be provided by CBP I alone. Cap binding complex activity can be partly separated from eIF4A activity on sucrose gradients, thus eIF4A provides a function that is distinct from cap binding complex activity. The results indicate that eIF4A can be physically separated from the cap binding complex without affecting the ability of the remaining structure to function in an in vitro translation system. They suggest that the eIF4A-free complex may provide a function that is not a property of either CBP I or of eIF4A, but may be a property of p220.     

p53 at the crossroad of DNA replication and ribosome biogenesis stress pathways

Despite several decades of intense research focused on understanding function(s) and disease-associated malfunction of p53, there is no sign of any “mid-life crisis” in this rapidly advancing area of biomedicine. Firmly established as the hub of cellular stress responses and tumor suppressor targeted in most malignancies, p53’s many talents continue to surprise us, providing not only fresh insights into cell and organismal biology, but also new avenues to cancer treatment. Among the most fruitful lines of p53 research in recent years have been the discoveries revealing the multifaceted roles of p53-centered pathways in the fundamental processes of DNA replication and ribosome biogenesis (RiBi), along with cellular responses to replication and RiBi stresses, two intertwined areas of cell (patho)physiology that we discuss in this review. Here, we first provide concise introductory notes on the canonical roles of p53, the key interacting proteins, downstream targets and post-translational modifications involved in p53 regulation. We then highlight the emerging involvement of p53 as a key component of the DNA replication Fork Speed Regulatory Network and the mechanistic links of p53 with cellular checkpoint responses to replication stress (RS), the driving force of cancer-associated genomic instability. Next, the tantalizing, yet still rather foggy functional crosstalk between replication and RiBi (nucleolar) stresses is considered, followed by the more defined involvement of p53-mediated monitoring of the multistep process of RiBi, including the latest updates on the RPL5/RPL11/5 S rRNA-MDM2-p53-mediated Impaired Ribosome Biogenesis Checkpoint (IRBC) pathway and its involvement in tumorigenesis. The diverse defects of RiBi and IRBC that predispose and/or contribute to severe human pathologies including developmental syndromes and cancer are then outlined, along with examples of promising small-molecule-based strategies to therapeutically target the RS- and particularly RiBi- stress-tolerance mechanisms to which cancer cells are addicted due to their aberrant DNA replication, repair, and proteo-synthesis demands. Subject terms: Cancer, Cell biology     

The ATPase and helicase activities of Prp43p are stimulated by the G‐patch protein Pfa1p during yeast ribosome biogenesis

Prp43p is a RNA helicase required for pre‐mRNA splicing and for the synthesis of large and small ribosomal subunits. The molecular functions and modes of regulation of Prp43p during ribosome biogenesis remain unknown. We demonstrate that the G‐patch protein Pfa1p, a component of pre‐40S pre‐ribosomal particles, directly interacts with Prp43p. We also show that lack of Gno1p, another G‐patch protein associated with Prp43p, specifically reduces Pfa1p accumulation, whereas it increases the levels of the pre‐40S pre‐ribosomal particle component Ltv1p. Moreover, cells lacking Pfa1p and depleted for Ltv1p show strong 20S pre‐rRNA accumulation in the cytoplasm and reduced levels of 18S rRNA. Finally, we demonstrate that Pfa1p stimulates the ATPase and helicase activities of Prp43p. Truncated Pfa1p variants unable to fully stimulate the activity of Prp43p fail to complement the 20S pre‐rRNA processing defect of Δpfa1 cells depleted for Ltv1p. Our results strongly suggest that stimulation of ATPase/helicase activities of Prp43p by Pfa1p is required for efficient 20S pre‐rRNA‐to‐18S rRNA conversion.     

Rrp5p, a trans-acting factor in yeast ribosome biogenesis, is an RNA-binding protein with a pronounced preference for U-rich sequences

Rrp5p is a trans-acting factor important for biogenesis of both the 40S and 60S subunit of the Saccharomyces cerevisiae ribosome. The protein contains 12 tandemly repeated S1 RNA binding motifs in its N-terminal region, suggesting the ability to interact directly with the pre-rRNA. In vitro binding studies, using immunopurified Rrp5p and in vitro transcribed, 32P-UTP-labeled RNA fragments, revealed that Rrp5p is a general RNA-binding protein with a strong preference for single-stranded sequences rich in uridines. Co-immunoprecipitation studies in yeast cells expressing ProtA-tagged Rrp5p showed that the protein is still associated with pre-ribosomal particles containing 27SA2 pre-rRNA but not with particles containing the 27SB precursor. Thus, Rrp5p appears to dissociate from the 66S pre-ribosome upon or immediately after further processing of 27SA2 pre-rRNA, suggesting the presence of (an) important binding site(s) within the 3'-terminal portion of ITS1. The location of these possible binding site(s) was further delimited using rrp2-1 mutant cells, which accumulate the 5'-extended 5.8S pre-rRNA species. The results indicate that association of Rrp5p with the pre-ribosome is abolished upon removal of a 30-nt region downstream from site A2, which contains two short, single-stranded U stretches. Sequence comparison shows that only the most 5' of these two U-rich stretches is conserved among yeast species whose ITS1 can functionally replace the S. cerevisiae spacer. The implications for the role of Rrp5p in yeast ribosome biogenesis are discussed.