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Pervasive transcription generates many unstable non-coding transcripts in budding yeast. The transcription of such noncoding RNAs, in particular antisense RNAs (asRNAs), has been shown in a few examples to repress the expression of the associated mRNAs. Yet, such mechanism is not known to commonly contribute to the regulation of a given class of genes. Using a mutant context that stabilised pervasive transcripts, we observed that the least expressed mRNAs during the exponential phase were associated with high levels of asRNAs. These asRNAs also overlapped their corresponding gene promoters with a much higher frequency than average. Interrupting antisense transcription of a subset of genes corresponding to quiescence-enriched mRNAs restored their expression. The underlying mechanism acts in cis and involves several chromatin modifiers. Our results convey that transcription interference represses up to 30% of the 590 least expressed genes, which includes 163 genes with quiescence-enriched mRNAs. We also found that pervasive transcripts constitute a higher fraction of the transcriptome in quiescence relative to the exponential phase, consistent with gene expression itself playing an important role to suppress pervasive transcription. Accordingly, the HIS1 asRNA, normally only present in quiescence, is expressed in exponential phase upon HIS1 mRNA transcription interruption.

International audience; Understanding how genes are regulated, decoding their "regulome", is one of the main challenges of the post-genomic era. Here, we describe the in vitro method we used to associate cis-regulatory sites with cognate trans-regulators by characterizing the DNA-binding specificity of the vast majority of yeast transcription factors using Protein Binding Microarrays. This approach can be implemented to any given organism.

International audience; Most homeodomains are unique within a genome, yet many are highly conserved across vast evolutionary distances, implying strong selection on their precise DNA-binding specificities. We determined the binding preferences of the majority (168) of mouse homeodomains to all possible 8base sequences, revealing rich and complex patterns of sequence specificity, and showing for the first time that there are at least 65 distinct homeodomain DNA-binding activities. We developed a computational system that successfully predicts binding sites for homeodomain proteins as distant from mouse as Drosophila and C. elegans, and we infer full 8-mer binding profiles for the majority of known animal homeodomains. Our results provide an unprecedented level of resolution in the analysis of this simple domain structure and suggest that variation in sequence recognition may be a factor in its functional diversity and evolutionary success.

Understanding how genes are regulated, decoding their "regulome", is one of the main challenges of the post-genomic era. Here, we describe the in vitro method we used to associate cis-regulatory sites with cognate trans-regulators by characterizing the DNA-binding specificity of the vast majority of yeast transcription factors using Protein Binding Microarrays. This approach can be implemented to any given organism.

SummarySince detection of an RNA molecule is the major criterion to define transcriptional activity, the fraction of the genome that is expressed is generally considered to parallel the complexity of the transcriptome. We show here that several supposedly silent intergenic regions in the genome of S. cerevisiae are actually transcribed by RNA polymerase II, suggesting that the expressed fraction of the genome is higher than anticipated. Surprisingly, however, RNAs originating from these regions are rapidly degraded by the combined action of the exosome and a new poly(A) polymerase activity that is defined by the Trf4 protein and one of two RNA binding proteins, Air1p or Air2p. We show that such a polyadenylation-assisted degradation mechanism is also responsible for the degradation of several Pol I and Pol III transcripts. Our data strongly support the existence of a posttranscriptional quality control mechanism limiting inappropriate expression of genetic information.

International audience; Ribosome biogenesis requires \textgreater300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C1 and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7(L)b within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.

Classifying proteins into subgroups with similar molecular function on the basis of sequence is an important step in deriving reliable functional annotations computationally. So far, however, available classification procedures have been evaluated against protein subgroups that are defined by experts using mainly qualitative descriptions of molecular function. Recently, in vitro DNA-binding preferences to all possible 8-nt DNA sequences have been measured for 178 mouse homeodomains using protein-binding microarrays, offering the unprecedented opportunity of evaluating the classification methods against quantitative measures of molecular function. To this end, we automatically derive homeodomain subtypes from the DNA-binding data and independently group the same domains using sequence information alone. We test five sequence-based methods, which use different sequence-similarity measures and algorithms to group sequences. Results show that methods that optimize the classification robustness reflect well the detailed functional specificity revealed by the experimental data. In some of these classifications, 73–83% of the subfamilies exactly correspond to, or are completely contained in, the function-based subtypes. Our findings demonstrate that certain sequence-based classifications are capable of yielding very specific molecular function annotations. The availability of quantitative descriptions of molecular function, such as DNA-binding data, will be a key factor in exploiting this potential in the future., Canadian Institutes of Health Research (MOP#82940), Sickkids Foundation, Ontario Research Fund, National Science Foundation (U.S.), National Human Genome Research Institute (U.S.) (R01 HG003985)

Members of the large ETS family of transcription factors (TFs) have highly similar DNA-binding domains (DBDs)—yet they have diverse functions and activities in physiology and oncogenesis. Some differences in DNA-binding preferences within this family have been described, but they have not been analysed systematically, and their contributions to targeting remain largely uncharacterized. We report here the DNA-binding profiles for all human and mouse ETS factors, which we generated using two different methods: a high-throughput microwell-based TF DNA-binding specificity assay, and protein-binding microarrays (PBMs). Both approaches reveal that the ETS-binding profiles cluster into four distinct classes, and that all ETS factors linked to cancer, ERG, ETV1, ETV4 and FLI1, fall into just one of these classes. We identify amino-acid residues that are critical for the differences in specificity between all the classes, and confirm the specificities in vivo using chromatin immunoprecipitation followed by sequencing (ChIP-seq) for a member of each class. The results indicate that even relatively small differences in in vitro binding specificity of a TF contribute to site selectivity in vivo.

Transcriptional Regulation Gets More Complicated Sequence preferences of DNA binding proteins are a primary mechanism by which cells interpret the genome. A central goal in genome biology is to identify regulatory sequences in the genome; however, few proteins' DNA binding specificities have been characterized comprehensively. Badis et al. (p. 1720 , published online 14 May) studied 104 known and predicted transcription factors (TFs), spanning 22 structural classes, in the mouse genome. While traditional models of TF binding sites are based on a single collection of highly similar DNA sequences, binding profiles were represented better by multiple motifs. Roughly half of the TFs recognized distinct primary and secondary motifs that are different from each other. At least some of these interaction modes appeared to be attributable to biophysically distinct protein conformations, adding to the complexity of transcriptional regulation.

Motivation: Recognition of specific DNA sequences is a central mechanism by which transcription factors (TFs) control gene expression. Many TF-binding preferences, however, are unknown or poorly characterized, in part due to the difficulty associated with determining their specificity experimentally, and an incomplete understanding of the mechanisms governing sequence specificity. New techniques that estimate the affinity of TFs to all possible k-mers provide a new opportunity to study DNA–protein interaction mechanisms, and may facilitate inference of binding preferences for members of a given TF family when such information is available for other family members. Results: We employed a new dataset consisting of the relative preferences of mouse homeodomains for all eight-base DNA sequences in order to ask how well we can predict the binding profiles of homeodomains when only their protein sequences are given. We evaluated a panel of standard statistical inference techniques, as well as variations of the protein features considered. Nearest neighbour among functionally important residues emerged among the most effective methods. Our results underscore the complexity of TF–DNA recognition, and suggest a rational approach for future analyses of TF families. Contact: t.hughes@utorotno.ca Supplementary information: Supplementary data are available at Bioinformatics online., Canadian Institutes of Health Research, Ontario Research Fund, National Institutes of Health (U.S.), National Human Genome Research Institute (U.S.)

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.

International audience; Ribosome biogenesis requires equimolar amounts of four rRNAs and all 79 ribosomal proteins (RP). Coordinated regulation of rRNA and RP synthesis by eukaryotic RNA polymerases (Pol) I, III, and II is a key requirement for growth control. Using a novel global genetic approach, we showed that the absence of Hmo1 becomes lethal when combined with mutations of components of either the RNA Pol II or Pol I transcription machineries, of specific RP, or of the TOR pathway. Hmo1 directly interacts with both the region transcribed by Pol I and a subset of RP gene promoters. Down-regulation of Hmo1 expression affects RP gene expression. Upon TORC1 inhibition, Hmo1 dissociates from ribosomal DNA (rDNA) and some RP gene promoters simultaneously. Finally, in the absence of Hmo1, TOR-dependent repression of RP genes is alleviated. Therefore, we show here that Saccharomyces cerevisiae Hmo1 is directly involved in coordinating rDNA transcription by Pol I and RP gene expression by Pol II under the control of the TOR pathway.

International audience; Conversion of uridines into pseudouridines (Psis) is the most frequent base modification in ribosomal RNAs (rRNAs). In eukaryotes, the pseudouridylation sites are specified by base-pairing with specific target sequences within H/ACA small nucleolar RNAs (snoRNAs). The yeast rRNAs harbor 44 Psis, but, when this work began, 15 Psis had completely unknown guide snoRNAs. This suggested that many snoRNAs remained to be discovered. To address this problem and further complete the snoRNA assignment to Psi sites, we identified the complete set of RNAs associated with the H/ACA snoRNP specific proteins Gar1p and Nhp2p by coupling TAP-tag purifications with genomic DNA microarrays experiments. Surprisingly, while we identified all the previously known H/ACA snoRNAs, we selected only three new snoRNAs. This suggested that most of the missing Psi guides were present in previously known snoRNAs but had been overlooked. We confirmed this hypothesis by systematically investigating the role of previously known, as well as of the newly identified snoRNAs, in specifying rRNA Psi sites and found all but one missing guide RNAs. During the completion of this work, another study, based on bioinformatic predictions, also reported the identification of most missing guide RNAs. Altogether, all Psi guides are now identified and we can tell that, in budding yeast, the 44 Psis are guided by 28 snoRNAs. Finally, aside from snR30, an atypical small RNA of heterogeneous length and at least one mRNA, all Gar1p and Nhp2p associated RNAs characterized by our work turned out to be snoRNAs involved in rRNA Psi specification.

International audience; Modulating the rate of mRNA degradation is a fast and efficient way to control gene expression. In a yeast strain deleted of EDC3, a component of the decapping machinery conserved in eukaryotes, the transcript coding the ribosomal protein Rps28b is specifically stabilized, as demonstrated by microarray and time course experiments. This stabilization results from the loss of RPS28B autoregulation, which occurs at the level of mRNA decay. Using mutants of the major deadenylase, we show that this regulation occurs at the level of decapping and bypasses deadenylation. Rps28b interacts with a conserved hairpin structure within the 3'UTR of its own mRNA and with components of the decapping machinery, including Edc3. We conclude that Rps28b, in the presence of Edc3, directly recruits the decapping machinery on its own mRNA. These findings show that specific modulation of the decapping efficiency on natural transcripts can control mRNA turnover.

International audience; Ribosomal RNAs contain a number of modified nucleotides. The most abundant nucleotide modifications found within rRNAs fall into two types: 2′-O-ribose methylations and pseudouridylations. In eukaryotes, small nucleolar guide RNAs, the snoRNAs that are the RNA components of the snoRNPs, specify the position of these modifications. The 2′-O-ribose methylations and pseudouridylations are guided by the box C/D and box H/ACA snoRNAs, respectively. The role of these modifications in rRNA remains poorly understood as no clear phenotype has yet been assigned to the absence of specific 2′-O-ribose methylations or pseudouridylations. Only very recently, a slight translation defect and perturbation of polysome profiles was reported in yeast for the absence of the Ψ at position 2919 within the LSU rRNA. Here we report the identification and characterization in yeast of a novel intronic H/ACA snoRNA that we called snR191 and that guides pseudouridylation at positions 2258 and 2260 in the LSU rRNA. Most interestingly, these two modified bases are the most conserved pseudouridines from bacteria to human in rRNA. The corresponding human snoRNA is hU19. We show here that, in yeast, the presence of this snoRNA, and hence, most likely, of the conserved pseudouridines it specifies, is not essential for viability but provides a growth advantage to the cell.

We have screened the genome of Saccharomyces cerevisiae for fragments that confer a growth-retardation phenotype when overexpressed in a multicopy plasmid with a tetracycline-regulatable (Tet-off) promoter. We selected 714 such fragments with a mean size of 700 base-pairs out of around 84,000 clones tested. These include 493 in-frame open reading frame fragments corresponding to 454 distinct genes (of which 91 are of unknown function), and 162 out-of-frame, antisense and intergenic genomic fragments, representing the largest collection of toxic inserts published so far in yeast.

International audience; Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway involved in many cellular pathways and crucial for telomere maintenance and embryo development. Core NMD factors Upf1, Upf2 and Upf3 are conserved from yeast to mammals, but a universal NMD model is lacking. We used affinity purification coupled with mass spectrometry and an improved data analysis protocol to obtain the first large-scale quantitative characterization of yeast NMD complexes in yeast (112 experiments). Unexpectedly, we identified two distinct complexes associated with Upf1: Detector (Upf1/2/3) and Effector. Effector contained the mRNA decapping enzyme, together with Nmd4 and Ebs1, two proteins that globally affected NMD and were critical for RNA degradation mediated by the Upf1 C-terminal helicase region. The fact that Nmd4 association to RNA was dependent on Detector components and the similarity between Nmd4/Ebs1 and mammalian Smg5-7 proteins suggest that NMD operates through successive Upf1-bound Detector and Effector complexes in other species. This model can be extended to accommodate steps that are missing in yeast, and thus serve for mechanistic studies of NMD in all eukaryotes. Running title: The Detector/Effector NMD model