Search

Identifying genes in the genomic context is central to a cell's ability to interpret the genome. Yet, in general, the signals used to define eukaryotic genes are poorly described. Here, we derived simple classifiers that identify where transcription will initiate and terminate using nucleic acid sequence features detectable by the yeast cell, which we integrate into a Unified Model (UM) that models transcription as a whole. The cis-elements that denote where transcription initiates function primarily through nucleosome depletion, and, using a synthetic promoter system, we show that most of these elements are sufficient to initiate transcription in vivo. Hrp1 binding sites are the major characteristic of terminators; these binding sites are often clustered in terminator regions and can terminate transcription bidirectionally. The UM predicts global transcript structure by modeling transcription of the genome using a hidden Markov model whose emissions are the outputs of the initiation and termination classifiers. We validated the novel predictions of the UM with available RNA-seq data and tested it further by directly comparing the transcript structure predicted by the model to the transcription generated by the cell for synthetic DNA segments of random design. We show that the UM identifies transcription start sites more accurately than the initiation classifier alone, indicating that the relative arrangement of promoter and terminator elements influences their function. Our model presents a concrete description of how the cell defines transcript units, explains the existence of nongenic transcripts, and provides insight into genome evolution.

The term "transcriptional network" refers to the mechanism(s) that underlies coordinated expression of genes, typically involving transcription factors (TFs) binding to the promoters of multiple genes, and individual genes controlled by multiple TFs. A multitude of studies in the last two decades have aimed to map and characterize transcriptional networks in the yeast Saccharomyces cerevisiae. We review the methodologies and accomplishments of these studies, as well as challenges we now face. For most yeast TFs, data have been collected on their sequence preferences, in vivo promoter occupancy, and gene expression profiles in deletion mutants. These systematic studies have led to the identification of new regulators of numerous cellular functions and shed light on the overall organization of yeast gene regulation. However, many yeast TFs appear to be inactive under standard laboratory growth conditions, and many of the available data were collected using techniques that have since been improved. Perhaps as a consequence, comprehensive and accurate mapping among TF sequence preferences, promoter binding, and gene expression remains an open challenge. We propose that the time is ripe for renewed systematic efforts toward a complete mapping of yeast transcriptional regulatory mechanisms.

Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology, and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors, including the CAF-1 complex, Spt10, and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning, and these data provide a valuable resource for future studies., Competing Interests: The authors have declared that no competing interests exist.

The yeast Saccharomyces cerevisiae is a prevalent system for the analysis of transcriptional networks. As a result, multiple DNA-binding sequence specificities (motifs) have been derived for most yeast transcription factors (TFs). However, motifs from different studies are often inconsistent with each other, making subsequent analyses complicated and confusing. Here, we have created YeTFaSCo (The Yeast Transcription Factor Specificity Compendium, http://yetfasco.ccbr.utoronto.ca/), an extensive collection of S. cerevisiae TF specificities. YeTFaSCo differs from related databases by being more comprehensive (including 1709 motifs for 256 proteins or protein complexes), and by evaluating the motifs using multiple objective quality metrics. The metrics include correlation between motif matches and ChIP-chip data, gene expression patterns, and GO terms, as well as motif agreement between different studies. YeTFaSCo also features an index of 'expert-curated' motifs, each associated with a confidence assessment. In addition, the database website features tools for motif analysis, including a sequence scanning function and precomputed genome-browser tracks of motif occurrences across the entire yeast genome. Users can also search the database for motifs that are similar to a query motif.

Background: A gene network's capacity to process information, so as to bind past events to future actions, depends on its structure and logic. From previous and new microarray measurements in Saccharomyces cerevisiae following gene deletions and overexpressions, we identify a core gene regulatory network (GRN) of functional interactions between 328 genes and the transfer functions of each gene. Inferred connections are verified by gene enrichment., Results: We find that this core network has a generalized clustering coefficient that is much higher than chance. The inferred Boolean transfer functions have a mean p-bias of 0.41, and thus similar amounts of activation and repression interactions. However, the distribution of p-biases differs significantly from what is expected by chance that, along with the high mean connectivity, is found to cause the core GRN of S. cerevisiae's to have an overall sensitivity similar to critical Boolean networks. In agreement, we find that the amount of information propagated between nodes in finite time series is much higher in the inferred core GRN of S. cerevisiae than what is expected by chance., Conclusions: We suggest that S. cerevisiae is likely to have evolved a core GRN with enhanced information propagation among its genes.

Cwc21 (complexed with Cef1 protein 21) is a 135 amino acid yeast protein that shares homology with the N-terminal domain of human SRm300/SRRM2, a large serine/arginine-repeat protein shown previously to associate with the splicing coactivator and 3'-end processing stimulatory factor, SRm160. Proteomic analysis of spliceosomal complexes has suggested a role for Cwc21 and SRm300 at the core of the spliceosome. However, specific functions for these proteins have remained elusive. In this report, we employ quantitative genetic interaction mapping, mass spectrometry of tandem affinity-purified complexes, and microarray profiling to investigate genetic, physical, and functional interactions involving Cwc21. Combined data from these assays support multiple roles for Cwc21 in the formation and function of splicing complexes. Consistent with a role for Cwc21 at the core of the spliceosome, we observe strong genetic, physical, and functional interactions with Isy1, a protein previously implicated in the first catalytic step of splicing and splicing fidelity. Together, the results suggest multiple functions for Cwc21/SRm300 in the splicing process, including an important role in the activation of splicing in association with Isy1.

The development and application of genomic reagents and techniques has fuelled progress in our understanding of regulatory networks that control gene expression in eukaryotic cells. However, a full description of the network of regulator-gene interactions that determine global gene expression programs remains elusive and will require systematic genetic as well as biochemical assays. Here, we describe a functional genomics approach that combines reporter technology, genome-wide array-based reagents and high-throughput imaging to discover new regulators controlling gene expression patterns in Saccharomyces cerevisiae. Our strategy utilizes the synthetic genetic array (SGA) method to systematically introduce promoter-GFP (green fluorescent protein) reporter constructs along with a control promoter-RFP (red fluorescent protein) gene into the array of approximately 4500 viable yeast deletion mutants. Fluorescence intensities from each reporter are assayed from individual colonies arrayed on solid agar plates using a scanning fluorimager and the ratio of GFP to RFP intensity reveals deletion mutants that cause differential GFP expression. We are exploiting this screening approach to construct a detailed map describing the interplay of regulators controlling the eukaryotic cell cycle. The method is extensible to any transcription factor or signalling pathway for which an appropriate reporter gene can be devised.

Nucleosome organization is critical for gene regulation. In living cells this organization is determined by multiple factors, including the action of chromatin remodellers, competition with site-specific DNA-binding proteins, and the DNA sequence preferences of the nucleosomes themselves. However, it has been difficult to estimate the relative importance of each of these mechanisms in vivo, because in vivo nucleosome maps reflect the combined action of all influencing factors. Here we determine the importance of nucleosome DNA sequence preferences experimentally by measuring the genome-wide occupancy of nucleosomes assembled on purified yeast genomic DNA. The resulting map, in which nucleosome occupancy is governed only by the intrinsic sequence preferences of nucleosomes, is similar to in vivo nucleosome maps generated in three different growth conditions. In vitro, nucleosome depletion is evident at many transcription factor binding sites and around gene start and end sites, indicating that nucleosome depletion at these sites in vivo is partly encoded in the genome. We confirm these results with a micrococcal nuclease-independent experiment that measures the relative affinity of nucleosomes for approximately 40,000 double-stranded 150-base-pair oligonucleotides. Using our in vitro data, we devise a computational model of nucleosome sequence preferences that is significantly correlated with in vivo nucleosome occupancy in Caenorhabditis elegans. Our results indicate that the intrinsic DNA sequence preferences of nucleosomes have a central role in determining the organization of nucleosomes in vivo.

The sequence specificity of DNA-binding proteins is the primary mechanism by which the cell recognizes genomic features. Here, we describe systematic determination of yeast transcription factor DNA-binding specificities. We obtained binding specificities for 112 DNA-binding proteins representing 19 distinct structural classes. One-third of the binding specificities have not been previously reported. Several binding sequences have striking genomic distributions relative to transcription start sites, supporting their biological relevance and suggesting a role in promoter architecture. Among these are Rsc3 binding sequences, containing the core CGCG, which are found preferentially approximately 100 bp upstream of transcription start sites. Mutation of RSC3 results in a dramatic increase in nucleosome occupancy in hundreds of proximal promoters containing a Rsc3 binding element, but has little impact on promoters lacking Rsc3 binding sequences, indicating that Rsc3 plays a broad role in targeting nucleosome exclusion at yeast promoters.

Previous studies in Saccharomyces cerevisiae have demonstrated that cryptic promoters within coding regions activate transcription in particular mutants. We have performed a comprehensive analysis of cryptic transcription in order to identify factors that normally repress cryptic promoters, to determine the amount of cryptic transcription genome-wide, and to study the potential for expression of genetic information by cryptic transcription. Our results show that a large number of factors that control chromatin structure and transcription are required to repress cryptic transcription from at least 1,000 locations across the S. cerevisiae genome. Two results suggest that some cryptic transcripts are translated. First, as expected, many cryptic transcripts contain an ATG and an open reading frame of at least 100 codons. Second, several cryptic transcripts are translated into proteins. Furthermore, a subset of cryptic transcripts tested is transiently induced in wild-type cells following a nutritional shift, suggesting a possible physiological role in response to a change in growth conditions. Taken together, our results demonstrate that, during normal growth, the global integrity of gene expression is maintained by a wide range of factors and suggest that, under altered genetic or physiological conditions, the expression of alternative genetic information may occur.

Hsp90 is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes. In this study, we identify a function for the chaperone in RNA processing and maintenance. This functionality of Hsp90 involves two recently identified interactors of the chaperone: Tah1 and Pih1/Nop17. Tah1 is a small protein containing tetratricopeptide repeats, whereas Pih1 is found to be an unstable protein. Tah1 and Pih1 bind to the essential helicases Rvb1 and Rvb2 to form the R2TP complex, which we demonstrate is required for the correct accumulation of box C/D small nucleolar ribonucleoproteins. Together with the Tah1 cofactor, Hsp90 functions to stabilize Pih1. As a consequence, the chaperone is shown to affect box C/D accumulation and maintenance, especially under stress conditions. Hsp90 and R2TP proteins are also involved in the proper accumulation of box H/ACA small nucleolar RNAs.

We present the first complete high-resolution map of nucleosome occupancy across the whole Saccharomyces cerevisiae genome, identifying over 70,000 positioned nucleosomes occupying 81% of the genome. On a genome-wide scale, the persistent nucleosome-depleted region identified previously in a subset of genes demarcates the transcription start site. Both nucleosome occupancy signatures and overall occupancy correlate with transcript abundance and transcription rate. In addition, functionally related genes can be clustered on the basis of the nucleosome occupancy patterns observed at their promoters. A quantitative model of nucleosome occupancy indicates that DNA structural features may account for much of the global nucleosome occupancy.

Mutations impacting specific stages of cell growth and division have provided a foundation for dissecting mechanisms that underlie cell cycle progression. We have undertaken an objective examination of the yeast cell cycle through flow cytometric analysis of DNA content in TetO(7) promoter mutant strains representing 75% of all essential yeast genes. More than 65% of the strains displayed specific alterations in DNA content, suggesting that reduced function of an essential gene in most cases impairs progression through a specific stage of the cell cycle. Because of the large number of essential genes required for protein biosynthesis, G1 accumulation was the most common phenotype observed in our analysis. In contrast, relatively few mutants displayed S-phase delay, and most of these were defective in genes required for DNA replication or nucleotide metabolism. G2 accumulation appeared to arise from a variety of defects. In addition to providing a global view of the diversity of essential cellular processes that influence cell cycle progression, these data also provided predictions regarding the functions of individual genes: we identified four new genes involved in protein trafficking (NUS1, PHS1, PGA2, PGA3), and we found that CSE1 and SMC4 are important for DNA replication.

Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.

Biogenesis of the small and large ribosomal subunits requires modification, processing, and folding of pre-rRNA to yield mature rRNA. Here, we report that efficient biogenesis of both small- and large-subunit rRNAs requires the DEAH box ATPase Prp43p, a pre-mRNA splicing factor. By steady-state analysis, a cold-sensitive prp43 mutant accumulates 35S pre-rRNA and depletes 20S, 27S, and 7S pre-rRNAs, precursors to the small- and large-subunit rRNAs. By pulse-chase analysis, the prp43 mutant is defective in the formation of 20S and 27S pre-rRNAs and in the accumulation of 18S and 25S mature rRNAs. Wild-type Prp43p immunoprecipitates pre-rRNAs and mature rRNAs, indicating a direct role in ribosome biogenesis. The Prp43p-Q423N mutant immunoprecipitates 27SA2 pre-rRNA threefold more efficiently than the wild type, suggesting a critical role for Prp43p at the earliest stages of large-subunit biogenesis. Consistent with an early role for Prp43p in ribosome biogenesis, Prp43p immunoprecipitates the majority of snoRNAs; further, compared to the wild type, the prp43 mutant generally immunoprecipitates the snoRNAs more efficiently. In the prp43 mutant, the snoRNA snR64 fails to methylate residue C2337 in 27S pre-rRNA, suggesting a role in snoRNA function. We propose that Prp43p promotes recycling of snoRNAs and biogenesis factors during pre-rRNA processing, similar to its recycling role in pre-mRNA splicing. The dual function for Prp43p in the cell raises the possibility that ribosome biogenesis and pre-mRNA splicing may be coordinately regulated.

The nature of synthetic genetic interactions involving essential genes (those required for viability) has not been previously examined in a broad and unbiased manner. We crossed yeast strains carrying promoter-replacement alleles for more than half of all essential yeast genes to a panel of 30 different mutants with defects in diverse cellular processes. The resulting genetic network is biased toward interactions between functionally related genes, enabling identification of a previously uncharacterized essential gene (PGA1) required for specific functions of the endoplasmic reticulum. But there are also many interactions between genes with dissimilar functions, suggesting that individual essential genes are required for buffering many cellular processes. The most notable feature of the essential synthetic genetic network is that it has an interaction density five times that of nonessential synthetic genetic networks, indicating that most yeast genetic interactions involve at least one essential gene.

Esf2p is the Saccharomyces cerevisiae homolog of mouse ABT1, a protein previously identified as a putative partner of the TATA-element binding protein. However, large-scale studies have indicated that Esf2p is primarily localized to the nucleolus and that it physically associates with pre-rRNA processing factors. Here, we show that Esf2p-depleted cells are defective for pre-rRNA processing at the early nucleolar cleavage sites A0 through A2 and consequently are inhibited for 18S rRNA synthesis. Esf2p was stably associated with the 5' external transcribed spacer (ETS) and the box C+D snoRNA U3, as well as additional box C+D snoRNAs and proteins enriched within the small-subunit (SSU) processome/90S preribosomes. Esf2p colocalized on glycerol gradients with 90S preribosomes and slower migrating particles containing 5' ETS fragments. Strikingly, upon Esf2p depletion, chromatin spreads revealed that SSU processome assembly and compaction are inhibited and glycerol gradient analysis showed that U3 remains associated within 90S preribosomes. This suggests that in the absence of proper SSU processome assembly, early pre-rRNA processing is inhibited and U3 is not properly released from the 35S pre-rRNAs. The identification of ABT1 in a large-scale analysis of the human nucleolar proteome indicates that its role may also be conserved in mammals.

We used a microarray containing probes that tile all known yeast noncoding RNAs (ncRNAs) to investigate RNA biogenesis on a global scale. The microarray verified a general loss of Box C/D snoRNAs in the TetO7-BCD1 mutant, which had previously been shown for only a handful of snoRNAs. We also monitored the accumulation of improperly processed flank sequences of pre-RNAs in strains depleted for known RNA nucleases, including RNase III, Dbr1p, Xrn1p, Rat1p and components of the exosome and RNase P complexes. Among the hundreds of aberrant RNA processing events detected, two novel substrates of Rnt1p (the RUF1 and RUF3 snoRNAs) were identified. We also identified a relationship between tRNA 5' end processing and tRNA splicing, processes that were previously thought to be independent. This analysis demonstrates the applicability of microarray technology to the study of global analysis of ncRNA synthesis and provides an extensive directory of processing events mediated by yeast ncRNA processing enzymes.

The SAGA complex is a multisubunit protein complex involved in transcriptional regulation in Saccharomyces cerevisiae. SAGA combines proteins involved in interactions with DNA-bound activators and TATA-binding protein (TBP), as well as enzymes for histone acetylation (Gcn5) and histone deubiquitylation (Ubp8). We recently showed that H2B ubiquitylation and Ubp8-mediated deubiquitylation are both required for transcriptional activation. For this study, we investigated the interaction of Ubp8 with SAGA. Using mutagenesis, we identified a putative zinc (Zn) binding domain within Ubp8 as being critical for the association with SAGA. The Zn binding domain is required for H2B deubiquitylation and for growth on media requiring Ubp8's function in gene activation. Furthermore, we identified an 11-kDa subunit of SAGA, Sgf11, and showed that it is required for the Ubp8 association with SAGA and for H2B deubiquitylation. Different approaches indicated that the functions of Ubp8 and Sgf11 are related and separable from those of other components of SAGA. In particular, the profiles of Ubp8 and Sgf11 deletions were remarkably similar in microarray analyses and synthetic genetic interactions and were distinct from those of the Spt3 and Spt8 subunits of SAGA, which are involved in TBP regulation. These data indicate that Ubp8 and Sgf11 likely represent a new functional module within SAGA that is involved in gene regulation through H2B deubiquitylation.

Using a microarray that tiles all known yeast non-coding RNAs, we compared RNA from wild-type cells with RNA from mutants encoding known and putative RNA modifying enzymes. We show that at least five types of RNA modification (dihydrouridine, m1G, m2(2)G, m1A and m6(2)A) catalyzed by 10 different enzymes (Trm1p, Trm5, Trm10p, Dus1p-Dus4p, Dim1p, Gcd10p and Gcd14p) can be detected by virtue of differential hybridization to oligonucleotides on the array that are complementary to the modified sites. Using this approach, we identified a previously undetected m1A modification in GlnCTG tRNA, the formation of which is catalyzed by the Gcd10/Gcd14 complex. complex.

A major objective in post-genome research is to fully understand the transcriptional control of each gene and the targets of each transcription factor. In yeast, large-scale experimental and computational approaches have been applied to identify co-regulated genes, cis regulatory elements, and transcription factor DNA binding sites in vivo. Methods for modeling and predicting system behavior, and for reconciling discrepancies among data types, are being explored. The results indicate that a complete and comprehensive yeast transcriptional network will ultimately be achieved.

Genome sequencing provides complete parts lists of organisms. This presents the obvious challenge of determining how each gene contributes to the life of the organism. This task seems increasingly feasible; however, progress to date suggests that increased interaction between systematic efforts and individual investigators will be critical to completing the encyclopedia of the yeast cell.

NuA4, the only essential histone acetyltransferase complex in Saccharomyces cerevisiae, acetylates the N-terminal tails of histones H4 and H2A. Affinity purification of NuA4 revealed the presence of three previously undescribed subunits, Vid21/Eaf1/Ydr359c, Swc4/Eaf2/Ygr002c, and Eaf7/Ynl136w. Experimental analyses revealed at least two functionally distinct sets of polypeptides in NuA4: (i) Vid21 and Yng2, and (ii) Eaf5 and Eaf7. Vid21 and Yng2 are required for bulk histone H4 acetylation and are functionally linked to the histone H2A variant Htz1 and the Swr1 ATPase complex (SWR-C) that assembles Htz1 into chromatin, whereas Eaf5 and Eaf7 have a different, as yet undefined, role. Mutations in Htz1, the SWR-C, and NuA4 cause defects in chromosome segregation that are consistent with genetic interactions we have observed between the genes encoding these proteins and genes encoding kinetochore components. Because SWR-C-dependent recruitment of Htz1 occurs in both transcribed and centromeric regions, a NuA4/SWR-C/Htz1 pathway may regulate both transcription and centromere function in S. cerevisiae.

Using DNA microarrays, we compared global transcript stability profiles following chemical inhibition of transcription to rpb1-1 (a temperature-sensitive allele of yeast RNA polymerase II). Among the five inhibitors tested, the effects of thiolutin and 1,10-phenanthroline were most similar to rpb1-1. A comparison to various microarray data already in the literature revealed similarity between mRNA stability profiles and the transcriptional response to stresses such as heat shock, consistent with the fact that the general stress response includes a transient shutoff of general mRNA transcription. Genes encoding factors involved in rRNA synthesis and ribosome assembly, which are often observed to be coordinately down-regulated in yeast microarray data, were among the least stable transcripts. We examined the effects of deletions of genes encoding deadenylase components Ccr4p and Pan2p and putative RNA-binding proteins Pub1p and Puf4p on the genome-wide pattern of mRNA stability after inhibition of transcription by chemicals and/or heat stress. This examination showed that Ccr4p, the major yeast mRNA deadenylase, contributes to the degradation of transcripts encoding both ribosomal proteins and rRNA synthesis and ribosome assembly factors and mediates a large part of the transcriptional response to heat stress. Pan2p and Puf4p also contributed to the degradation rate of these mRNAs following transcriptional shutoff, while Pub1p preferentially stabilized transcripts encoding ribosomal proteins. Our results indicate that the abundance of ribosome biogenesis factors is controlled at the level of mRNA stability.

Dihydrouridine is a highly abundant modified nucleoside found widely in tRNAs of eubacteria, eukaryotes, and some archaea. In cytoplasmic tRNA of Saccharomyces cerevisiae, dihydrouridine occurs exclusively at positions 16, 17, 20, 20A, 20B, and 47. Here we show that the known dihydrouridine synthases Dus1p and Dus2p and two previously uncharacterized homologs, Dus3p (encoded by YLR401c) and Dus4p (YLR405w), are required for all of the dihydrouridine modification of cytoplasmic tRNAs in S. cerevisiae. We have mapped the in vivo position specificity of the four Dus proteins, by three complementary approaches: determination of the molar ratio of dihydrouridine in purified tRNAs from different dus mutants; microarray analysis of a large number of tRNAs based on differential hybridization of uridine and dihydrouridine-containing tRNAs to the complementary oligonucleotides; and the development and use of a novel dihydrouridine mapping technique, employing primer extension. We show that each of the four Dus proteins has a distinct position specificity: Dus1p for U(16) and U(17), Dus2p for U(20), Dus3p for U(47), and Dus4p for U(20a) and U(20b).

We report that Esf1p (Ydr365cp), an essential, evolutionarily conserved nucleolar protein, is required for the biogenesis of 18S rRNA in Saccharomyces cerevisiae. Depletion of Esf1p resulted in delayed processing of 35S precursor and a striking loss of 18S rRNA. Esf1p physically associated with ribosomal proteins and proteins involved in 18S rRNA biogenesis. Consistent with its role in 18S rRNA biogenesis, Esf1p also physically associated with U3 and U14 snoRNAs, but did not appear to be a core component of the SSU processome. These data indicate that Esf1p plays a direct role in early pre-rRNA processing.

A remarkably large collection of evolutionarily conserved proteins has been implicated in processing of noncoding RNAs and biogenesis of ribonucleoproteins. To better define the physical and functional relationships among these proteins and their cognate RNAs, we performed 165 highly stringent affinity purifications of known or predicted RNA-related proteins from Saccharomyces cerevisiae. We systematically identified and estimated the relative abundance of stably associated polypeptides and RNA species using a combination of gel densitometry, protein mass spectrometry, and oligonucleotide microarray hybridization. Ninety-two discrete proteins or protein complexes were identified comprising 489 different polypeptides, many associated with one or more specific RNA molecules. Some of the pre-rRNA-processing complexes that were obtained are discrete sub-complexes of those previously described. Among these, we identified the IPI complex required for proper processing of the ITS2 region of the ribosomal RNA primary transcript. This study provides a high-resolution overview of the modular topology of noncoding RNA-processing machinery.

Bioactive compounds can be valuable research tools and drug leads, but it is often difficult to identify their mechanism of action or cellular target. Here we investigate the potential for integration of chemical-genetic and genetic interaction data to reveal information about the pathways and targets of inhibitory compounds. Taking advantage of the existing complete set of yeast haploid deletion mutants, we generated drug-hypersensitivity (chemical-genetic) profiles for 12 compounds. In addition to a set of compound-specific interactions, the chemical-genetic profiles identified a large group of genes required for multidrug resistance. In particular, yeast mutants lacking a functional vacuolar H(+)-ATPase show multidrug sensitivity, a phenomenon that may be conserved in mammalian cells. By filtering chemical-genetic profiles for the multidrug-resistant genes and then clustering the compound-specific profiles with a compendium of large-scale genetic interaction profiles, we were able to identify target pathways or proteins. This method thus provides a powerful means for inferring mechanism of action.

In Saccharomyces cerevisiae, polarized morphogenesis is critical for bud site selection, bud development, and cell separation. The latter is mediated by Ace2p transcription factor, which controls the daughter cell-specific expression of cell separation genes. Recently, a set of proteins that include Cbk1p kinase, its binding partner Mob2p, Tao3p (Pag1p), and Hym1p were shown to regulate both Ace2p activity and cellular morphogenesis. These proteins seem to form a signaling network, which we designate RAM for regulation of Ace2p activity and cellular morphogenesis. To find additional RAM components, we conducted genetic screens for bilateral mating and cell separation mutants and identified alleles of the PAK-related kinase Kic1p in addition to Cbk1p, Mob2p, Tao3p, and Hym1p. Deletion of each RAM gene resulted in a loss of Ace2p function and caused cell polarity defects that were distinct from formin or polarisome mutants. Two-hybrid and coimmunoprecipitation experiments reveal a complex network of interactions among the RAM proteins, including Cbk1p-Cbk1p, Cbk1p-Kic1p, Kic1p-Tao3p, and Kic1p-Hym1p interactions, in addition to the previously documented Cbk1p-Mob2p and Cbk1p-Tao3p interactions. We also identified a novel leucine-rich repeat-containing protein Sog2p that interacts with Hym1p and Kic1p. Cells lacking Sog2p exhibited the characteristic cell separation and cell morphology defects associated with perturbation in RAM signaling. Each RAM protein localized to cortical sites of growth during both budding and mating pheromone response. Hym1p was Kic1p- and Sog2p-dependent and Sog2p and Kic1p were interdependent for localization, indicating a close functional relationship between these proteins. Only Mob2p and Cbk1p were detectable in the daughter cell nucleus at the end of mitosis. The nuclear localization and kinase activity of the Mob2p-Cbk1p complex were dependent on all other RAM proteins, suggesting that Mob2p-Cbk1p functions late in the RAM network. Our data suggest that the functional architecture of RAM signaling is similar to the S. cerevisiae mitotic exit network and Schizosaccharomyces pombe septation initiation network and is likely conserved among eukaryotes.

Small, cell permeable, and target-specific chemical ligands are highly valuable, not only as therapeutics but also as research tools. The synthesis, identification and characterization of these compounds is often a difficult task. The straightforward genetics of the budding yeast Saccharomyces cerevisiae, and the high degree of conservation of basic cellular processes between yeast and higher organisms makes yeast an excellent tool for drug development studies, particularly in regards to anticancer and anti-fungal drug discovery. Recent advances in yeast functional genomics and proteomics studies are changing the field of yeast research. Many of these new technologies are readily applicable to drug target identification and other aspects of drug discovery. This review will focus on current genetic, genomic, and proteomic methodologies in S. cerevisiae that have the potential to be useful in drug discovery and target validation.

Advanced genetic techniques, along with the high degree of conservation of basic cellular processes, have made the yeast Saccharomyces cerevisiae a valuable system for identification of new drug targets, target-based and non-target-based drug screening, and detailed analysis of the cellular effects of drugs. Yeast also presents a convenient system for antifungal drug discovery because it is closely related to Candida albicans, a major human pathogen. Many yeast genes remain poorly characterized, and most of the sophisticated techniques in yeast have been in widespread use less than a decade - a period shorter than the typical cycle from target identification to marketing approval for a new drug. It is likely that most of the benefits of yeast in discovery and development of therapeutic compounds have yet to be realized.

Genome sequencing has led to the discovery of tens of thousands of potential new genes. Six years after the sequencing of the well-studied yeast Saccharomyces cerevisiae and the discovery that its genome encodes approximately 6,000 predicted proteins, more than 2,000 have not yet been characterized experimentally, and determining their functions seems far from a trivial task. One crucial constraint is the generation of useful hypotheses about protein function. Using a new approach to interpret microarray data, we assign likely cellular functions with confidence values to these new yeast proteins. We perform extensive genome-wide validations of our predictions and offer visualization methods for exploration of the large numbers of functional predictions. We identify potential new members of many existing functional categories including 285 candidate proteins involved in transcription, processing and transport of non-coding RNA molecules. We present experimental validation confirming the involvement of several of these proteins in ribosomal RNA processing. Our methodology can be applied to a variety of genomics data types and organisms.

Ascertaining the impact of uncharacterized perturbations on the cell is a fundamental problem in biology. Here, we describe how a single assay can be used to monitor hundreds of different cellular functions simultaneously. We constructed a reference database or "compendium" of expression profiles corresponding to 300 diverse mutations and chemical treatments in S. cerevisiae, and we show that the cellular pathways affected can be determined by pattern matching, even among very subtle profiles. The utility of this approach is validated by examining profiles caused by deletions of uncharacterized genes: we identify and experimentally confirm that eight uncharacterized open reading frames encode proteins required for sterol metabolism, cell wall function, mitochondrial respiration, or protein synthesis. We also show that the compendium can be used to characterize pharmacological perturbations by identifying a novel target of the commonly used drug dyclonine.

Expression profiling using DNA microarrays holds great promise for a variety of research applications, including the systematic characterization of genes discovered by sequencing projects. To demonstrate the general usefulness of this approach, we recently obtained expression profiles for nearly 300 Saccharomyces cerevisiae deletion mutants. Approximately 8% of the mutants profiled exhibited chromosome-wide expression biases, leading to spurious correlations among profiles. Competitive hybridization of genomic DNA from the mutant strains and their isogenic parental wild-type strains showed they were aneuploid for whole chromosomes or chromosomal segments. Expression profile data published by several other laboratories also suggest the use of aneuploid strains. In five separate cases, the extra chromosome harboured a close homologue of the deleted gene; in two cases, a clear growth advantage for cells acquiring the extra chromosome was demonstrated. Our results have implications for interpreting whole-genome expression data, particularly from cells known to suffer genomic instability, such as malignant or immortalized cells.

EST1, EST2, EST3 and TLC1 function in a single pathway for telomere replication in the yeast Saccharomyces cerevisiae [1] [2], as would be expected if these genes all encode components of the same complex. Est2p, the reverse transcriptase protein subunit, and TLC1, the templating RNA, are subunits of the catalytic core of yeast telomerase [3] [4] [5]. In contrast, mutations in EST1, EST3 or CDC13 eliminate telomere replication in vivo [1] [6] [7] [8] but are dispensable for in vitro telomerase catalytic activity [2] [9]. Est1p and Cdc13p, as components of telomerase and telomeric chromatin, respectively, cooperate to recruit telomerase to the end of the chromosome [7] [10]. However, Est3p has not yet been biochemically characterized and thus its specific role in telomere replication is unclear. We show here that Est3p is a stable component of the telomerase holoenzyme and furthermore, association of Est3p with the enzyme requires an intact catalytic core. As predicted for a telomerase subunit, fusion of Est3p to the high affinity Cdc13p telomeric DNA binding domain greatly increases access of telomerase to the telomere. Est1p is also tightly associated with telomerase; however, Est1p is capable of forming a stable TLC1-containing complex even in the absence of Est2p or Est3p. Yeast telomerase therefore contains a minimum of three Est proteins for which there is both in vivo and in vitro evidence for their role in telomere replication as subunits of the telomerase complex.

The CDC13 gene of Saccharomyces cerevisiae is required both to protect telomeric DNA and to ensure proper function of yeast telomerase in vivo. We have previously demonstrated that Cdc13p has a high affinity single-strand telomeric DNA binding activity, although the primary amino acid sequence of Cdc13p has no previously characterized DNA binding motifs. We report here mapping of the Cdc13 DNA binding domain by a combination of proteolysis mapping and deletion cloning. The DNA binding domain maps to residues 557-694 of the 924-amino acid Cdc13 polypeptide, within the most basic region of Cdc13p. A slightly larger version of this domain can be efficiently expressed in Escherichia coli as a soluble small protein, with DNA binding properties comparable to those of the full-length protein. A single amino acid missense mutation within this domain results in thermolabile DNA binding and conditional lethality in yeast, consistent with the prediction that DNA binding should be essential for CDC13 function. These results show that Cdc13p contains a discrete substructure responsible for DNA binding and should facilitate structural characterization of this telomere binding protein.

Genome-wide transcript profiling was used to monitor signal transduction during yeast pheromone response. Genetic manipulations allowed analysis of changes in gene expression underlying pheromone signaling, cell cycle control, and polarized morphogenesis. A two-dimensional hierarchical clustered matrix, covering 383 of the most highly regulated genes, was constructed from 46 diverse experimental conditions. Diagnostic subsets of coexpressed genes reflected signaling activity, cross talk, and overlap of multiple mitogen-activated protein kinase (MAPK) pathways. Analysis of the profiles specified by two different MAPKs-Fus3p and Kss1p-revealed functional overlap of the filamentous growth and mating responses. Global transcript analysis reflects biological responses associated with the activation and perturbation of signal transduction pathways.

Genetic selections were used to find peptides that inhibit biological pathways in budding yeast. The peptides were presented inside cells as peptamers, surface loops on a highly expressed and biologically inert carrier protein, a catalytically inactive derivative of staphylococcal nuclease. Peptamers that inhibited the pheromone signaling pathway, transcriptional silencing, and the spindle checkpoint were isolated. Putative targets for the inhibitors were identified by a combination of two-hybrid analysis and genetic dissection of the target pathways. This analysis identified Ydr517w as a component of the spindle checkpoint and reinforced earlier indications that Ste50 has both positive and negative roles in pheromone signaling. Analysis of transcript arrays showed that the peptamers were highly specific in their effects, which suggests that they may be useful reagents in organisms that lack sophisticated genetics as well as for identifying components of existing biological pathways that are potential targets for drug discovery.

Telomerase is a specialized reverse transcriptase consisting of both RNA and protein components. Previous characterization of yeast telomerase function in vivo identified four EST (for ever shorter telomeres) genes that, when mutated, result in the phenotypes expected for a defect in telomerase. Consistent with this genetic prediction, the EST2 gene has recently been shown to encode the catalytic component of telomerase. Using an in vitro assay, we show here that telomerase activity is present in extracts prepared from yeast strains carrying est1-Delta, est3-Delta, and cdc13-2(est) mutations. Therefore, while these three genes are necessary for telomerase function in vivo, they do not encode components essential for core catalytic activity. When Est2p, the one EST gene product found to be essential for catalytic activity, was immunoprecipitated from extracts, the telomerase RNA subunit was also specifically precipitated, supporting the conclusion that these two components are in a stable complex.

We have recently completed a large mutant screen designed to identify new mutants of Saccharomyces cerevisiae with a telomerase-like defect. From this screen; 22 mutants were identified that mapped to three genes, called EST1, EST2 and EST3, as well as a novel EST-like mutation in a fourth gene, previously identified as CDC13. Mutations in each of these genes give rise to phenotypes that are indistinguishable from those observed when TLC1, encoding the yeast telomerase RNA, is deleted. In addition, genetic analysis indicates that all four genes function in the same pathway for telomere replication as defined by TLC1, the one known component of telomerase. This indicates that these genes encode factors that are essential in vivo for telomerase function. Genetic and biochemical analyses have shown that EST1 and CDC13 encode single-stranded telomeric DNA-binding proteins, suggesting that these two proteins may function to mediate access of telomerase to the end of the telomere.

The CDC13 gene has previously been implicated in the maintenance of telomere integrity in Saccharomyces cerevisiae. With the use of two classes of mutations, here it is shown that CDC13 has two discrete roles at the telomere. The cdc13-2est mutation perturbs a function required in vivo for telomerase regulation but not in vitro for enzyme activity, whereas cdc13-1ts defines a separate essential role at the telomere. In vitro, purified Cdc13p binds to single-strand yeast telomeric DNA. Therefore, Cdc13p is a telomere-binding protein required to protect the telomere and mediate access of telomerase to the chromosomal terminus.