A New System for Comparative Functional Genomics of Saccharomyces Yeasts

Whole-genome sequencing, particularly in fungi, has progressed at a tremendous rate. More difficult, however, is experimental testing of the inferences about gene function that can be drawn from comparative sequence analysis alone. We present a genome-wide functional characterization of a sequenced but experimentally understudied budding yeast, Saccharomyces bayanus var. uvarum (henceforth referred to as S. bayanus), allowing us to map changes over the 20 million years that separate this organism from S. cerevisiae. We first created a suite of genetic tools to facilitate work in S. bayanus. Next, we measured the gene-expression response of S. bayanus to a diverse set of perturbations optimized using a computational approach to cover a diverse array of functionally relevant biological responses. The resulting data set reveals that gene-expression patterns are largely conserved, but significant changes may exist in regulatory networks such as carbohydrate utilization and meiosis. In addition to regulatory changes, our approach identified gene functions that have diverged. The functions of genes in core pathways are highly conserved, but we observed many changes in which genes are involved in osmotic stress, peroxisome biogenesis, and autophagy. A surprising number of genes specific to S. bayanus respond to oxidative stress, suggesting the organism may have evolved under different selection pressures than S. cerevisiae. This work expands the scope of genome-scale evolutionary studies from sequence-based analysis to rapid experimental characterization and could be adopted for functional mapping in any lineage of interest. Furthermore, our detailed characterization of S. bayanus provides a valuable resource for comparative functional genomics studies in yeast.     

Molecular phylogeny of pectinase genes <Emphasis Type="Italic">PGU</Emphasis> in the yeast genus <Emphasis Type="Italic">Saccharomyces</Emphasis>

Using yeast genome databases and literature data, phylogenetic analysis of pectinase PGU genes from 112 Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and the hybrid taxon S. pastorianus (syn. S. carlsbergensis) was carried out. A superfamily of divergent PGU genes was found. Natural interspecies transfer of the PGU gene both from S. cerevisiae to S. bayanus and from S. paradoxus to S. cerevisiae may, however, occur. Within the Saccharomyces species, identity of the PGU nucleotide sequences was 98.8–100% for S. cerevisiae, 86.1–95.7% for S. bayanus (var. uvarum), 94–98.3% for S. kudriavzevii, and 96.8–100% for S. paradoxus/S. cariocanus. For the first time, a family of polymeric PGU1b, PGU2b, PGU3b and PGU4b genes is documented for the yeast S. bayanus var. uvarum, a variety important for winemaking.     

On the Complexity of the Saccharomyces bayanus Taxon: Hybridization and Potential Hybrid Speciation

Although the genus Saccharomyces has been thoroughly studied, some species in the genus has not yet been accurately resolved; an example is S. bayanus, a taxon that includes genetically diverse lineages of pure and hybrid strains. This diversity makes the assignation and classification of strains belonging to this species unclear and controversial. They have been subdivided by some authors into two varieties (bayanus and uvarum), which have been raised to the species level by others. In this work, we evaluate the complexity of 46 different strains included in the S. bayanus taxon by means of PCR-RFLP analysis and by sequencing of 34 gene regions and one mitochondrial gene. Using the sequence data, and based on the S. bayanus var. bayanus reference strain NBRC 1948, a hypothetical pure S. bayanus was reconstructed for these genes that showed alleles with similarity values lower than 97% with the S. bayanus var. uvarum strain CBS 7001, and of 99–100% with the non S. cerevisiae portion in S. pastorianus Weihenstephan 34/70 and with the new species S. eubayanus. Among the S. bayanus strains under study, different levels of homozygosity, hybridization and introgression were found; however, no pure S. bayanus var. bayanus strain was identified. These S. bayanus hybrids can be classified into two types: homozygous (type I) and heterozygous hybrids (type II), indicating that they have been originated by different hybridization processes. Therefore, a putative evolutionary scenario involving two different hybridization events between a S. bayanus var. uvarum and unknown European S. eubayanus-like strains can be postulated to explain the genomic diversity observed in our S. bayanus var. bayanus strains.     

Systematic Planning of Genome-Scale Experiments in Poorly Studied Species

Genome-scale datasets have been used extensively in model organisms to screen for specific candidates or to predict functions for uncharacterized genes. However, despite the availability of extensive knowledge in model organisms, the planning of genome-scale experiments in poorly studied species is still based on the intuition of experts or heuristic trials. We propose that computational and systematic approaches can be applied to drive the experiment planning process in poorly studied species based on available data and knowledge in closely related model organisms. In this paper, we suggest a computational strategy for recommending genome-scale experiments based on their capability to interrogate diverse biological processes to enable protein function assignment. To this end, we use the data-rich functional genomics compendium of the model organism to quantify the accuracy of each dataset in predicting each specific biological process and the overlap in such coverage between different datasets. Our approach uses an optimized combination of these quantifications to recommend an ordered list of experiments for accurately annotating most proteins in the poorly studied related organisms to most biological processes, as well as a set of experiments that target each specific biological process. The effectiveness of this experiment- planning system is demonstrated for two related yeast species: the model organism Saccharomyces cerevisiae and the comparatively poorly studied Saccharomyces bayanus. Our system recommended a set of S. bayanus experiments based on an S. cerevisiae microarray data compendium. In silico evaluations estimate that less than 10% of the experiments could achieve similar functional coverage to the whole microarray compendium. This estimation was confirmed by performing the recommended experiments in S. bayanus, therefore significantly reducing the labor devoted to characterize the poorly studied genome. This experiment-planning framework could readily be adapted to the design of other types of large-scale experiments as well as other groups of organisms.     

Influence of choice of yeasts on volatile fermentation-derived compounds, colour and phenolics composition in Cabernet Sauvignon wine

Wine colour, phenolics and volatile fermentation-derived composition are the quintessential elements of a red wine. Many viticultural and winemaking factors contribute to wine aroma and colour with choice of yeast strain being a crucial factor. Besides the traditional Saccharomyces species S. cerevisiae, S. bayanus and several Saccharomyces interspecific hybrids are able to ferment grape juice to completion. This study examined the diversity in chemical composition, including phenolics and fermentation-derived volatile compounds, of an Australian Cabernet Sauvignon due to the use of different Saccharomyces strains. Eleven commercially available Saccharomyces strains were used in this study; S. cerevisiae (7), S. bayanus (2) and interspecific Saccharomyces hybrids (2). The eleven Cabernet Sauvignon wines varied greatly in their chemical composition. Nine yeast strains completed alcoholic fermentation in 19?days; S. bayanus AWRI 1375 in 26?days, and S. cerevisiae AWRI 1554 required 32?days. Ethanol concentrations varied in the final wines (12.7?C14.2?%). The two S. bayanus strains produced the most distinct wines, with the ability to metabolise malic acid, generate high glycerol concentrations and distinctive phenolic composition. Saccharomyces hybrid AWRI 1501 and S. cerevisiae AWRI 1554 and AWRI 1493 also generated distinctive wines. This work demonstrates that the style of a Cabernet Sauvignon can be clearly modulated by choice of commercially available wine yeast.     

Pure and Mixed Genetic Lines of Saccharomyces bayanus and Saccharomyces pastorianus and Their Contribution to the Lager Brewing Strain Genome

The yeast species Saccharomyces bayanus and Saccharomyces pastorianus are of industrial importance since they are involved in the production process of common beverages such as wine and lager beer; however, they contain strains whose variability has been neither fully investigated nor exploited in genetic improvement programs. We evaluated this variability by using PCR-restriction fragment length polymorphism analysis of 48 genes and partial sequences of 16. Within these two species, we identified “pure” strains containing a single type of genome and “hybrid” strains that contained portions of the genomes from the “pure” lines, as well as alleles termed “Lager” that represent a third genome commonly associated with lager brewing strains. The two pure lines represent S. uvarum and S. bayanus, the latter a novel group of strains that may be of use in strain improvement programs. Hybrid lines identified include (i) S. cerevisiae/S. bayanus/Lager, (ii) S. bayanus/S. uvarum/Lager, and (iii) S. cerevisiae/S. bayanus/S. uvarum/Lager. The genome of the lager strains may have resulted from chromosomal loss, replacement, or rearrangement within the hybrid genetic lines. This study identifies brewing strains that could be used as novel genetic sources in strain improvement programs and provides data that can be used to generate a model of how naturally occurring and industrial hybrid strains may have evolved.     

Identification and polymorphism of pectinase genes <Emphasis Type="Italic">PGU</Emphasis> in the <Emphasis Type="Italic">Saccharomyces bayanus</Emphasis> complex

Pectinase (endo-polygalacturonase) is the key enzyme splitting plant pectin. The corresponding single gene PGU1 is documented for the yeast S. cerevisiae. On the basis of phylogenetic analysis of the PGU nucleotide sequence available in the GenBank, a family of divergent PGU genes is found in the species complex S. bayanus: S. bayanus var. uvarum, S. eubayanus, and hybrid taxon S. pastorianus. The PGU genes have different chromosome localization.     

New family of pectinase genes <Emphasis Type="Italic">PGU1</Emphasis>b–<Emphasis Type="Italic">PGU3b</Emphasis> of the pectinolytic yeast <Emphasis Type="Italic">Saccharomyces bayanus</Emphasis> var. <Emphasis Type="Italic">uvarum</Emphasis>

Using yeast genome databases and literature data, we have conducted a phylogenetic analysis of pectinase PGU genes from Saccharomyces strains assigned to the biological species S. arboricola, S. bayanus (var. uvarum), S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, S. paradoxus, and hybrid taxon S. pastorianus (syn. S. carlsbergensis). Single PGU genes were observed in all Saccharomyces species, except S. bayanus. The superfamily of divergent PGU genes has been documented in S. bayanus var. uvarum for the first time. Chromosomal localization of new PGU1b, PGU2b, and PGU3b genes in the yeast S. bayanus var. uvarum has been determined by molecular karyotyping and Southern hybridization.     

Characterization of Chromosome Stability in Diploid,Polyploid and Hybrid Yeast Cells

Chromosome instability is a key component of cancer progression and many heritable diseases. Understanding why some chromosomes are more unstable than others could provide insight into understanding genome integrity. Here we systematically investigate the spontaneous chromosome loss for all sixteen chromosomes in Saccharomyces cerevisiae in order to elucidate the mechanisms underlying chromosome instability. We observed that the stability of different chromosomes varied more than 100-fold. Consistent with previous studies on artificial chromosomes, chromosome loss frequency was negatively correlated to chromosome length in S. cerevisiae diploids, triploids and S. cerevisiae-S. bayanus hybrids. Chromosome III, an equivalent of sex chromosomes in budding yeast, was found to be the most unstable chromosome among all cases examined. Moreover, similar instability was observed in chromosome III of S. bayanus, a species that diverged from S. cerevisiae about 20 million years ago, suggesting that the instability is caused by a conserved mechanism. Chromosome III was found to have a highly relaxed spindle checkpoint response in the genome. Using a plasmid stability assay, we found that differences in the centromeric sequence may explain certain aspects of chromosome instability. Our results reveal that even under normal conditions, individual chromosomes in a genome are subject to different levels of pressure in chromosome loss (or gain).     

Evolutionary Genomics of Transposable Elements in Saccharomyces cerevisiae

Saccharomyces cerevisiae is one of the premier model systems for studying the genomics and evolution of transposable elements. The availability of the S. cerevisiae genome led to unprecedented insights into its five known transposable element families (the LTR retrotransposons Ty1-Ty5) in the years shortly after its completion. However, subsequent advances in bioinformatics tools for analysing transposable elements and the recent availability of genome sequences for multiple strains and species of yeast motivates new investigations into Ty evolution in S. cerevisiae. Here we provide a comprehensive phylogenetic and population genetic analysis of all Ty families in S. cerevisiae based on a systematic re-annotation of Ty elements in the S288c reference genome. We show that previous annotation efforts have underestimated the total copy number of Ty elements for all known families. In addition, we identify a new family of Ty3-like elements related to the S. paradoxus Ty3p which is composed entirely of degenerate solo LTRs. Phylogenetic analyses of LTR sequences identified three families with short-branch, recently active clades nested among long branch, inactive insertions (Ty1, Ty3, Ty4), one family with essentially all recently active elements (Ty2) and two families with only inactive elements (Ty3p and Ty5). Population genomic data from 38 additional strains of S. cerevisiae show that the majority of Ty insertions in the S288c reference genome are fixed in the species, with insertions in active clades being predominantly polymorphic and insertions in inactive clades being predominantly fixed. Finally, we use comparative genomic data to provide evidence that the Ty2 and Ty3p families have arisen in the S. cerevisiae genome by horizontal transfer. Our results demonstrate that the genome of a single individual contains important information about the state of TE population dynamics within a species and suggest that horizontal transfer may play an important role in shaping the genomic diversity of transposable elements in unicellular eukaryotes.     

Deciphering the Hybridisation History Leading to the Lager Lineage Based on the Mosaic Genomes of Saccharomyces bayanus Strains NBRC1948 and CBS380T

Saccharomyces bayanus is a yeast species described as one of the two parents of the hybrid brewing yeast S. pastorianus. Strains CBS380^T and NBRC1948 have been retained successively as pure-line representatives of S. bayanus. In the present study, sequence analyses confirmed and upgraded our previous finding: S. bayanus type strain CBS380^T harbours a mosaic genome. The genome of strain NBRC1948 was also revealed to be mosaic. Both genomes were characterized by amplification and sequencing of different markers, including genes involved in maltotriose utilization or genes detected by array-CGH mapping. Sequence comparisons with public Saccharomyces spp. nucleotide sequences revealed that the CBS380^T and NBRC1948 genomes are composed of: a predominant non-cerevisiae genetic background belonging to S. uvarum, a second unidentified species provisionally named S. lagerae, and several introgressed S. cerevisiae fragments. The largest cerevisiae-introgressed DNA common to both genomes totals 70kb in length and is distributed in three contigs, cA, cB and cC. These vary in terms of length and presence of MAL31 or MTY1 (maltotriose-transporter gene). In NBRC1948, two additional cerevisiae-contigs, cD and cE, totaling 12kb in length, as well as several smaller cerevisiae fragments were identified. All of these contigs were partially detected in the genomes of S. pastorianus lager strains CBS1503 (S. monacensis) and CBS1513 (S. carlsbergensis) explaining the noticeable common ability of S. bayanus and S. pastorianus to metabolize maltotriose. NBRC1948 was shown to be inter-fertile with S. uvarum CBS7001. The cross involving these two strains produced F1 segregants resembling the strains CBS380^T or NRRLY-1551. This demonstrates that these S. bayanus strains were the offspring of a cross between S. uvarum and a strain similar to NBRC1948. Phylogenies established with selected cerevisiae and non-cerevisiae genes allowed us to decipher the complex hybridisation events linking S. lagerae/S. uvarum/S. cerevisiae with their hybrid species, S. bayanus/pastorianus.     

Authentication of ATCC strains in theSaccharomyces cerevisiae complex by PCR fingerprinting

Restriction fragment length polymorphisms in the rDNA internal transcribed spacer region (ITS) of 18 yeast strains currently assigned toSaccharomyces cerevisiae, S. pastorianus, andS. bayanus were examined. Primers complementary to the ITS region were used to amplify the ITS rDNA by the polymerase chain reaction (PCR). The products were digested with 10 endonucleases and cluster analysis was used to generate a phenogram from the restriction fragment data. Three strains ofS. cerevisiae (ATCC 10609, 26250, and 66162) exhibited restriction patterns that were different from the type strain but identical to those of theS. bayanus-S. pastorianus cluster. In contrast,S. pastorianus (ATCC 76671) showed restriction profiles that were different from its type strain but were identical to the type strain ofS. cerevisiae (ATCC 18824). These results suggest that the three strains ofS. cerevisiae should be reassigned to eitherS. pastorianus orS. bayanus, and the strain ofS. pastorianus (ATCC 76671) should be reclassified asS. cerevisiae.     

Chimeric Genomes of Natural Hybrids of Saccharomyces cerevisiae and Saccharomyces kudriavzevii

Recently, a new type of hybrid resulting from the hybridization between Saccharomyces cerevisiae and Saccharomyces kudriavzevii was described. These strains exhibit physiological properties of potential biotechnological interest. A preliminary characterization of these hybrids showed a trend to reduce the S. kudriavzevii fraction of the hybrid genome. We characterized the genomic constitution of several wine S. cerevisiae × S. kudriavzevii strains by using a combined approach based on the restriction fragment length polymorphism analysis of gene regions, comparative genome hybridizations with S. cerevisiae DNA arrays, ploidy analysis, and gene dose determination by quantitative real-time PCR. The high similarity in the genome structures of the S. cerevisiae × S. kudriavzevii hybrids under study indicates that they originated from a single hybridization event. After hybridization, the hybrid genome underwent extensive chromosomal rearrangements, including chromosome losses and the generation of chimeric chromosomes by the nonreciprocal recombination between homeologous chromosomes. These nonreciprocal recombinations between homeologous chromosomes occurred in highly conserved regions, such as Ty long terminal repeats (LTRs), rRNA regions, and conserved protein-coding genes. This study supports the hypothesis that chimeric chromosomes may have been generated by a mechanism similar to the recombination-mediated chromosome loss acting during meiosis in Saccharomyces hybrids. As a result of the selective processes acting during fermentation, hybrid genomes maintained the S. cerevisiae genome but reduced the S. kudriavzevii fraction.The genus Saccharomyces consists of seven biological species: S. arboricolus, S. bayanus, S. cariocanus, S. cerevisiae, S. kudriavzevii, S. mikatae, and S. paradoxus (29, 59) and the partially allotetraploid species S. pastorianus (46, 58).The hybrid species S. pastorianus, restricted to lager brewing environments, arose from two or more natural hybridization events between S. cerevisiae and a S. bayanus-like yeast (7, 16, 28, 46). Recent studies of S. bayanus have also revealed the hybrid nature of certain strains of this species, which has subsequently been subdivided into two groups, S. bayanus var. bayanus, containing a variety of hybrid strains, and S. bayanus var. uvarum, also referred to as S. uvarum, that contains nonhybrid strains (45, 46).New hybrids of other species from the genus Saccharomyces have recently been described. Hybrid yeasts of S. cerevisiae and S. kudriavzevii have been characterized among wine (6, 20, 33) and brewing yeasts (21); even triple hybrids of S. cerevisiae, S. bayanus, and S. kudriavzevii have been identified (20, 41).The first natural Saccharomyces interspecific hybrid identified, the lager brewing yeast S. pastorianus (S. carlsbergensis) (42, 57), has become one of the most investigated types of yeast hybrids. The genome structure of these hybrids has been examined by competitive array comparative genome hybridization (aCGH) (5, 16, 28), complete genome sequencing (28), and PCR-restriction fragment length polymorphism (RFLP) analysis of 48 genes and partial sequences of 16 genes (46). The aCGH analyses of several S. pastorianus strains with S. cerevisiae-only DNA arrays (5, 28) revealed the presence of aneuploidies due to deletions of entire regions of the S. cerevisiae fraction of the hybrid genomes. A recent aCGH analysis of S. pastorianus strains with S. cerevisiae and S. bayanus DNA arrays (16) showed two groups of strains according to their genome structure and composition. These groups arose from two independent hybridization events, and each one is characterized by a reduction and an amplification of the S. cerevisiae genome fraction, respectively.The genetic characterization of the wine S. cerevisiae and S. kudriavzevii hybrids by restriction analysis of five nuclear genes located in different chromosomes, 5.8S-ITS rDNA region and the mitochondrial COX2 gene, revealed the presence of three types of hybrids in Swiss wines, thus indicating the presence of different hybrid genomes (20). In a recent study (21), we identified six new types of S. cerevisiae and S. kudriavzevii hybrids among brewing strains, which were compared to wine hybrids by a genetic characterization based on RFLP analysis of 35 protein-encoding genes. This analysis confirmed the presence of three different genome types among wine hybrids that contain putative chimeric chromosomes, probably generated by a recombination between homeologous chromosomes of different parental origins.The aim of the present study is to investigate the genome composition and structure of wine hybrids of S. cerevisiae and S. kudriavzevii. This has been achieved by a combined approach based on the RFLP analysis of 35 gene regions from our previous study, comparative genome hybridizations using S. cerevisiae DNA macroarrays, a ploidy analysis by flow cytometry, and gene dose determinations by quantitative real-time PCR. This multiple approach allowed us to confirm the presence of chimeric chromosomes and define the mechanisms involved in their origins.     

Molecular genetic diversity of the Saccharomyces yeasts in Taiwan: Saccharomyces arboricola, Saccharomyces cerevisiae and Saccharomyces kudriavzevii

Genetic hybridization, sequence and karyotypic analyses of natural Saccharomyces yeasts isolated in different regions of Taiwan revealed three biological species: Saccharomyces arboricola, Saccharomyces cerevisiae and Saccharomyces kudriavzevii. Intraspecies variability of the D1/D2 and ITS1 rDNA sequences was detected among S. cerevisiae and S. kudriavzevii isolates. According to molecular and genetic analyses, the cosmopolitan species S. cerevisiae and S. kudriavzevii contain local divergent populations in Taiwan, Malaysia and Japan. Six of the seven known Saccharomyces species are documented in East Asia: S. arboricola, S. bayanus, S. cerevisiae, S. kudriavzevii, S. mikatae, and S. paradoxus.     

Characterisation ofSaccharomyces cerevisiae genes encoding ribosomal protein YL6

We have characterised aSaccharomyces cerevisiae cDNA (cDNA13), originally isolated on the basis of the short half-life of the corresponding mRNA. We show here that its sequence is closely related to that of the genes encoding ribosomal proteins K37, KD4 and K5 ofSchizosaccharomyces pombe. ‘mRNA13’ also behaves like other mRNAs encoding ribosomal proteins, in that its abundance increases sharply when glucose is added to cells grown on ethanol (nutrient-up shift), and declines when cells are subjected to a mild heat-shock. Unspliced mRNA13 accumulates when cells bearing a temperature-sensitive splicing mutation are grown at the restrictive temperature. The gene(s) corresponding to cDNA13, like other ribosomal protein genes ofS. cerevisiae, thus contain an intron. Southern blot analysis indicates the presence of two separate loci related to cDNA13 in theS. cerevisiae genome. From the sequence of one of these, a complete polypeptide sequence was deduced. The first 40 amino acids are identical to those of YL6, aS. cerevisiae ribosomal protein characterised only by N-terminal protein sequence analysis. There is clear evidence within the genomic sequence for the predicted intron, and for elements similar to those that regulate expression of otherS. cerevisiae ribosomal protein genes.     

Molecular polymorphism of β-fructosidase SUC genes in the Saccharomyces yeasts

The molecular polymorphism of SUC genes that encode β-fructosidase has been investigated in the yeast genus Saccharomyces. We have determined the nucleotide sequences of subtelomeric SUC3, SUC5, SUC7, SUC8, SUC9, and SUC10 genes of S. cerevisiae and the SUCa gene of S. arboricola. Comparisons of the nucleotide sequences of all known SUC genes revealed the predominance of C → T transitions in the third codon position, which were silent. The amino acid sequences of β-fructosidases studied have identity of 88–100%. SUCa (S. arboricola) and SUCb (S. bayanus) proteins, which had amino acid identity with other SUC proteins of less than 92%, were the most divergent. It was determined that accumulation of the polymeric SUC genes takes place in industrial populations of S. cerevisiae, while the other Saccharomyces species (S. arboricola, S. bayanus, S. cariocanus, S. kudriavzevii, S. mikatae, and S. paradoxus) each harbor only one SUC gene. Subtelomeric repeats of β-fructosidase SUC genes could appear in the genome of S. cerevisiae under the effect of selection in the course of their domestication.     

Evolutionary Modeling and Prediction of Non-Coding RNAs in Drosophila

We performed benchmarks of phylogenetic grammar-based ncRNA gene prediction, experimenting with eight different models of structural evolution and two different programs for genome alignment. We evaluated our models using alignments of twelve Drosophila genomes. We find that ncRNA prediction performance can vary greatly between different gene predictors and subfamilies of ncRNA gene. Our estimates for false positive rates are based on simulations which preserve local islands of conservation; using these simulations, we predict a higher rate of false positives than previous computational ncRNA screens have reported. Using one of the tested prediction grammars, we provide an updated set of ncRNA predictions for D. melanogaster and compare them to previously-published predictions and experimental data. Many of our predictions show correlations with protein-coding genes. We found significant depletion of intergenic predictions near the 3′ end of coding regions and furthermore depletion of predictions in the first intron of protein-coding genes. Some of our predictions are colocated with larger putative unannotated genes: for example, 17 of our predictions showing homology to the RFAM family snoR28 appear in a tandem array on the X chromosome; the 4.5 Kbp spanned by the predicted tandem array is contained within a FlyBase-annotated cDNA.     

Global analysis of yeast RNA processing identifies new targets of RNase III and uncovers a link between tRNA 5' end processing and tRNA splicing

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.