MOESM8 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning

Additional file 8. Nucleosome positioning of genes with significant changes between wt and dst1∆. Genes were ordered by the number of nucleosomes that changed (in occupancy or fuzziness) between the wt and dst1∆. The nucleosomal profile of the top five genes is presented.

Cytoplasmic 5′-3′ exonuclease Xrn1p is also a genome-wide transcription factor in yeast

The 5′ to 3′ exoribonuclease Xrn1 is a large protein involved in cytoplasmatic mRNA degradation as a critical component of the major decaysome. Its deletion in the yeast Saccharomyces cerevisiae is not lethal, but it has multiple physiological effects. In a previous study, our group showed that deletion of all tested components of the yeast major decaysome, including XRN1, results in a decrease in the synthetic rate and an increase in half-life of most mRNAs in a compensatory manner. Furthermore, the same study showed that the all tested decaysome components are also nuclear proteins that bind to the 5′ region of a number of genes. In the present work, we show that disruption of Xrn1 activi…

Growth rate controls mRNA turnover in steady and non-steady states.

Gene expression has been investigated in relation with growth rate in the yeast Saccharomyces cerevisiae, following different experimental strategies. The expression of some specific gene functional categories increases or decreases with growth rate. Our recently published results have unveiled that these changes in mRNA concentration with growth depend on the relative alteration of mRNA synthesis and decay, and that, in addition to this gene-specific transcriptomic signature of growth, global mRNA turnover increases with growth rate. We discuss here these results in relation with other previous and concurrent publications, and we add new evidence which indicates that growth rate controls m…

Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning.

Background TFIIS stimulates RNA cleavage by RNA polymerase II and promotes the resolution of backtracking events. TFIIS acts in the chromatin context, but its contribution to the chromatin landscape has not yet been investigated. Co-transcriptional chromatin alterations include subtle changes in nucleosome positioning, like those expected to be elicited by TFIIS, which are elusive to detect. The most popular method to map nucleosomes involves intensive chromatin digestion by micrococcal nuclease (MNase). Maps based on these exhaustively digested samples miss any MNase-sensitive nucleosomes caused by transcription. In contrast, partial digestion approaches preserve such nucleosomes, but intr…

The distribution of active RNA polymerase II along the transcribed region is gene-specific and controlled by elongation factors.

In order to study the intragenic profiles of active transcription, we determined the relative levels of active RNA polymerase II present at the 3'- and 5'-ends of 261 yeast genes by run-on. The results obtained indicate that the 3'/5' run-on ratio varies among the genes studied by over 12 log(2) units. This ratio seems to be an intrinsic characteristic of each transcriptional unit and does not significantly correlate with gene length, G + C content or level of expression. The correlation between the 3'/5' RNA polymerase II ratios measured by run-on and those obtained by chromatin immunoprecipitation is poor, although the genes encoding ribosomal proteins present exceptionally low ratios in …

The relative importance of transcription rate, cryptic transcription and mRNA stability on shaping stress responses in yeast

It has been recently stated that stress-responding genes in yeast are enriched in cryptic transcripts and that this is the cause of the differences observed between mRNA amount and RNA polymerase occupancy profiles. Other studies have shown that such differences are mainly due to modulation of mRNA stabilities. Here we analyze the relationship between the presence of cryptic transcripts in genes and their stress response profiles. Despite some of the stress-responding gene groups being indeed enriched in specific classes of cryptic transcripts, we found no statistically significant evidence that cryptic transcription is responsible for the differences observed between mRNA and transcription…

Regulon-Specific Control of Transcription Elongation across the Yeast Genome

Transcription elongation by RNA polymerase II was often considered an invariant non-regulated process. However, genome-wide studies have shown that transcriptional pausing during elongation is a frequent phenomenon in tightly-regulated metazoan genes. Using a combination of ChIP-on-chip and genomic run-on approaches, we found that the proportion of transcriptionally active RNA polymerase II (active versus total) present throughout the yeast genome is characteristic of some functional gene classes, like those related to ribosomes and mitochondria. This proportion also responds to regulatory stimuli mediated by protein kinase A and, in relation to cytosolic ribosomal-protein genes, it is medi…

Xrn1 influence on gene transcription results from the combination of general effects on elongating RNA pol II and gene-specific chromatin configuration.

mRNA homoeostasis is favoured by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5ʹ exhibited significant alterations that were com…

Gene expression is circular: factors for mRNA degradation also foster mRNA synthesis.

SummaryMaintaining proper mRNA levels is a key aspect in the regulation of gene expression. The balance between mRNA synthesis and decay determines these levels. We demonstrate that most yeast mRNAs are degraded by the cytoplasmic 5′-to-3′ pathway (the “decaysome”), as proposed previously. Unexpectedly, the level of these mRNAs is highly robust to perturbations in this major pathway because defects in various decaysome components lead to transcription downregulation. Moreover, these components shuttle between the cytoplasm and the nucleus, in a manner dependent on proper mRNA degradation. In the nucleus, they associate with chromatin—preferentially ∼30 bp upstream of transcription start-sit…

MOESM11 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning

Additional file 11. Occupancy-versus-fuzziness changes of the TATA-like gene bodies and + 1 nucleosomes. Heat maps of the difference between the mutant dst1∆ and the wt in fuzziness versus occupancy for the gene body nucleosomes of the TATA-like genes (A), and for the + 1 nucleosome (defined as that between the TSS and 200 bp downstream) of each gene (B).

MOESM7 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning

Additional file 7. Nucleotide composition of the sequence of the TATA and TATA-like genes. A) Frequency of each nucleotide in the TATA (red) and TATA-like genes (blue) at each position in relation to the TSS. B) The average nucleotide frequency in the promoter (− 500 to − 100) and the gene body (50–500) of the TATA and TATA-like genes. A Student’s t test was applied to compare the TATA and TATA-like genes. S indicates that the difference is significant (p 0.001).

The ribosome assembly gene network is controlled by the feedback regulation of transcription elongation

Ribosome assembly requires the concerted expression of hundreds of genes, which are transcribed by all three nuclear RNA polymerases. Transcription elongation involves dynamic interactions between RNA polymerases and chromatin. We performed a synthetic lethal screening in Saccharomyces cerevisiae with a conditional allele of SPT6, which encodes one of the factors that facilitates this process. Some of these synthetic mutants corresponded to factors that facilitate pre-rRNA processing and ribosome biogenesis. We found that the in vivo depletion of one of these factors, Arb1, activated transcription elongation in the set of genes involved directly in ribosome assembly. Under these depletion c…

MOESM6 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning

Additional file 6. A metagene analysis to compare the sequencing data before and after the correction in TATA genes versus TATA-like genes A) The metagene analysis of the chromatin (blue before the correction, red afterward) and the naked DNA signals (green) around the pAS in the TATA (left panel) and TATA-like genes (right panel). Genes were scaled to the same length and then aligned to their pAS. B) Genes were divided into quartiles according to their transcription rate [45] and then further subdivided into TATA or TATA-like genes. All the resulting eight groups were scaled and aligned to their TSS. The chromatin signal before and after correction is shown.

Genome-wide studies of mRNA synthesis and degradation in eukaryotes

In recent years, the use of genome-wide technologies has revolutionized the study of eukaryotic transcription producing results for thousands of genes at every step of mRNA life. The statistical analyses of the results for a single condition, different conditions, different transcription stages, or even between different techniques, is outlining a totally new landscape of the eukaryotic transcription process. Although most studies have been conducted in the yeast Saccharomyces cerevisiae as a model cell, others have also focused on higher eukaryotes, which can also be comparatively analyzed. The picture which emerges is that transcription is a more variable process than initially suspected,…

MOESM1 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning

Additional file 1. Overview of the method. A) A diagram with the main protocol steps is shown. The fragments to be sequenced were isolated from an ethidium bromide-stained gel (see the example in the figure). The naked DNA samples were visually matched to the chromatin samples by choosing those with a similar maximum fragment size (arrow). Then, the mononucleosome-sized fragments (squares) were isolated. B) The chromatin (blue and red) and naked DNA signals (green) over the STL1 gene are shown as examples of the results, analyzed by qPCR. The chromatin data are presented before (blue) and after (red) the naked DNA correction. C) The naked DNA signal in the STL1 gene from different Saccharom…

Eukaryotic mRNA decay: methodologies, pathways, and links to other stages of gene expression.

mRNA concentration depends on the balance between transcription and degradation rates. On both sides of the equilibrium, synthesis and degradation show, however, interesting differences that have conditioned the evolution of gene regulatory mechanisms. Here, we discuss recent genome-wide methods for determining mRNA half-lives in eukaryotes. We also review pre- and posttranscriptional regulons that coordinate the fate of functionally related mRNAs by using protein- or RNA-based trans factors. Some of these factors can regulate both transcription and decay rates, thereby maintaining proper mRNA homeostasis during eukaryotic cell life.

Homeostasis in the Central Dogma of molecular biology: the importance of mRNA instability

Cell survival requires the control of biomolecule concentration, i.e. biomolecules should approach homeostasis. With information-carrying macromolecules, the particular concentration variation ranges depend on each type: DNA is not buffered, but mRNA and protein concentrations are homeostatically controlled, which leads to the ribostasis and proteostasis concepts. In recent years, we have studied the particular features of mRNA ribostasis and proteostasis in the model organism S. cerevisiae. Here we extend this study by comparing published data from three other model organisms: E. coli, S. pombe and cultured human cells. We describe how mRNA ribostasis is less strict than proteostasis. A co…

Structural Characterization of Set1 RNA Recognition Motifs and their Role in Histone H3 Lysine 4 Methylation

Departament de Bioquimica iBiologia Molecular, Universitatde Valencia, C/Dr Moliner 50,46100, Burjassot, SpainThe yeast Set1 histone H3 lysine 4 (H3K4) methyltransferase contains, inaddition to its catalytic SET domain, a conserved RNA recognition motif(RRM1). We present here the crystal structure and the secondary structureassignment in solution of the Set1 RRM1. Although RRM1 has the expectedβαββαβ RRM-fold, it lacks the typical RNA-binding features of thesemodules. RRM1 is not able to bind RNA by itself in vitro, but a constructcombining RRM1 with a newly identified downstream RRM2 specificallybinds RNA. Invivo,H3K4 methylation isnot affectedbyapoint mutation inRRM2 that preserves Set1 s…

Nucleo-cytoplasmic shuttling of RNA-binding factors: mRNA buffering and beyond.

Gene expression is a highly regulated process that adapts RNAs and proteins content to the cellular context. Under steady-state conditions, mRNA homeostasis is robustly maintained by tight controls that act on both nuclear transcription and cytoplasmic mRNA stability. In recent years, it has been revealed that several RNA-binding proteins (RBPs) that perform functions in mRNA decay can move to the nucleus and regulate transcription. The RBPs involved in transcription can also travel to the cytoplasm and regulate mRNA degradation and/or translation. The multifaceted functions of these shuttling nucleo-cytoplasm RBPs have raised the possibility that they can act as mRNA metabolism coordinator…

A Gene-Specific Requirement for FACT during Transcription Is Related to the Chromatin Organization of the Transcribed Region

The FACT complex stimulates transcription elongation on nucleosomal templates. In vivo experiments also involve FACT in the reassembly of nucleosomes traversed by RNA polymerase II. Since several features of chromatin organization vary throughout the genome, we wondered whether FACT is equally required for all genes. We show in this study that the in vivo depletion of Spt16, one of the subunits of Saccharomyces cerevisiae FACT, strongly affects transcription of three genes, GAL1, PHO5, and Kluyveromyces lactis LAC4, which exhibit positioned nucleosomes at their transcribed regions. In contrast, showing a random nucleosome structure, YAT1 and Escherichia coli lacZ are only mildly influenced …

Cell volume homeostatically controls the rDNA repeat copy number and rRNA synthesis rate in yeast

AbstractThe adjustment of transcription and translation rates to variable needs is of utmost importance for the fitness and survival of living cells. We have previously shown that the global transcription rate for RNA polymerase II is regulated differently in cells presenting symmetrical or asymmetrical cell division. The budding yeast Saccharomyces cerevisiae adopts a particular strategy to avoid that the smaller daughter cells increase their total mRNA concentration with every generation. The global mRNA synthesis rate lowers with a growing cell volume, but global mRNA stability increases. In this paper, we address what the solution is to the same theoretical problem for the RNA polymeras…

Asymmetric cell division requires specific mechanisms for adjusting global transcription

Most cells divide symmetrically into two approximately identical cells. There are many examples, however, of asymmetric cell division that can generate sibling cell size differences. Whereas physical asymmetric division mechanisms and cell fate consequences have been investigated, the specific problem caused by asymmetric division at the transcription level has not yet been addressed. In symmetrically dividing cells the nascent transcription rate increases in parallel to cell volume to compensate it by keeping the actualmRNA synthesis rate constant. This cannot apply to the yeast Saccharomyces cerevisiae, where this mechanism would provoke a neverending increasing mRNA synthesis rate in sma…

A complete set of nascent transcription rates for yeast genes

The amount of mRNA in a cell is the result of two opposite reactions: transcription and mRNA degradation. These reactions are governed by kinetics laws, and the most regulated step for many genes is the transcription rate. The transcription rate, which is assumed to be exercised mainly at the RNA polymerase recruitment level, can be calculated using the RNA polymerase densities determined either by run-on or immunoprecipitation using specific antibodies. The yeast Saccharomyces cerevisiae is the ideal model organism to generate a complete set of nascent transcription rates that will prove useful for many gene regulation studies. By combining genomic data from both the GRO (Genomic Run-on) a…

The total mRNA concentration buffering system in yeast is global rather than gene-specific

Gene expression in eukaryotes does not follow a linear process from transcription to translation and mRNA degradation. Instead it follows a circular process in which cytoplasmic mRNA decay crosstalks with nuclear transcription. In many instances, this crosstalk contributes to buffer mRNA at a roughly constant concentration. Whether the mRNA buffering concept operates on the total mRNA concentration or at the gene-specific level, and if the mechanism to do so is a global or a specific one, remain unknown. Here we assessed changes in mRNA concentrations and their synthesis rates along the transcriptome of aneuploid strains of the yeast Saccharomyces cerevisiae. We also assessed mRNA concentra…

The cellular growth rate controls overall mRNA turnover, and modulates either transcription or degradation rates of particular gene regulons

We analyzed 80 different genomic experiments, and found a positive correlation between both RNA polymerase II transcription and mRNA degradation with growth rates in yeast. Thus, in spite of the marked variation in mRNA turnover, the total mRNA concentration remained approximately constant. Some genes, however, regulated their mRNA concentration by uncoupling mRNA stability from the transcription rate. Ribosome-related genes modulated their transcription rates to increase mRNA levels under fast growth. In contrast, mitochondria-related and stress-induced genes lowered mRNA levels by reducing mRNA stability or the transcription rate, respectively. We also detected these regulations within th…

MOESM3 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning

Additional file 3. Comparison with chemical mapping method. A) Center-to-center distance of the nearest nucleosome in: the raw data presented here against a chemical modification-based map [31] (blue line), the corrected data against the same reference map [31] (orange line), or the chemical modification-based map against a map that was generated by extensive digestion with MNase [12]. B) Cladogram showing the distance between the different maps mentioned in A.

MOESM5 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning

Additional file 5. A metagene analysis to compare the sequencing data before and after correction in different groups of genes. A) A 2D plot to compare the log10 signal intensity in the naked DNA sample and the GC content of fragments (normalized by subtracting the genomic average). Pearson’s correlation is shown (p

MOESM2 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning

Additional file 2. Metagene analysis of the chromatin and naked DNA signals. A, B) Genes were scaled to the same length and then aligned to their TSS or their pAS. All the genes in the yeast genome for which a TSS was available were considered. Zoom-in view of the data in Fig. 1a: A) closer to the TSS; B) closer to the pAS. C) Those genes whose pAS was at least 500 bp away from a TSS were selected, scaled to the same length, and represented as in B. D) Difference between the corrected and raw signals. Genes were scaled and aligned as in Fig. 1a, b. The Y-axis represents the logarithm of the p value of the difference. Two different curves are shown: one represents the positive difference val…

Chromatin-dependent regulation of RNA polymerases II and III activity throughout the transcription cycle

The particular behaviour of eukaryotic RNA polymerases along different gene regions and amongst distinct gene functional groups is not totally understood. To cast light onto the alternative active or backtracking states of RNA polymerase II, we have quantitatively mapped active RNA polymerases at a high resolution following a new biotin-based genomic run-on (BioGRO) technique. Compared with conventional profiling with chromatin immunoprecipitation, the analysis of the BioGRO profiles in Saccharomyces cerevisiae shows that RNA polymerase II has unique activity profiles at both gene ends, which are highly dependent on positioned nucleosomes. This is the first demonstration of the in vivo infl…

MOESM9 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning

Additional file 9. Nucleosome fuzziness in the wt and dst1∆. A) The metagene analysis of the fuzziness score of the wt (blue) and dst1∆ (red) nucleosomes around the TSS. Genes were scaled to the same length and then aligned to their TSS. B) The change in fuzziness score between the wt and dst1∆. Heat map of the fuzziness score of the gene body nucleosomes in the wt and dst1∆ mutant. Color represents density, which increases from blue to red. The red square highlights those nucleosomes below 40 in the wt and above > 40 in the mutant. C) The fuzziness score distribution of the nucleosomes in the gene bodies of the highly transcribed genes of the wt (blue) and dst1∆ (red). D) The fuzziness …

What do you mean by transcription rate?

mRNA synthesis in all organisms is performed by RNA polymerases, which work as nanomachines on DNA templates. The rate at which their product is made is an important parameter in gene expression. Transcription rate encompasses two related, yet different, concepts: the nascent transcription rate, which measures the in situ mRNA production by RNA polymerase, and the rate of synthesis of mature mRNA, which measures the contribution of transcription to the mRNA concentration. Both parameters are useful for molecular biologists, but they are not interchangeable and they are expressed in different units. It is important to distinguish when and where each one should be used. We propose that for fu…

MOESM10 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning

Additional file 10. Effect of the absence of TFIIS on the expression of the different types of genes. A) Scatter plot of the nascent transcription rate of each gene in the wt (X-axis) and in dst1∆ (Y-axis). B) Diagram showing the relationship between the group of genes with a weaker GRO signal in dst1∆ compared to the wt (TFIIS-dependent) in the TATA-containing genes and TATA-like genes. The TATA-containing genes are overrepresented in the TFIIS-dependent genes (hypergeometric test, p = 0.006), while the TATA-like genes are under-represented (hypergeometric test, p = 0.028). C) The Bio-GRO signals of the RP genes in the wt and dst1∆. After classification, genes were aligned to their TSS.

Corrigendum to “External conditions inversely change the RNA polymerase II elongation rate and density in yeast” [Biochim. Biophys. Acta 1829/11 (2013) 1248–1255]

Protein Interactions within the Set1 Complex and Their Roles in the Regulation of Histone 3 Lysine 4 Methylation

Set1 is the catalytic subunit and the central component of the evolutionarily conserved Set1 complex (Set1C) that methylates histone 3 lysine 4 (H3K4). Here we have determined protein/protein interactions within the complex and related the substructure to function. The loss of individual Set1C subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation, and distribution of H3K4 methylation along active genes. The complex requires Set1, Swd1, and Swd3 for integrity, and Set1 amount is greatly reduced in the absence of the Swd1-Swd3 heterodimer. Bre2 and Sdc1 also form a heteromeric subunit, which requires the SET domain for interaction with the complex, and Sdc…

External conditions inversely change the RNA polymerase II elongation rate and density in yeast.

Elongation speed is a key parameter in RNA polymerase II (RNA pol II) activity. It affects the transcription rate, while it is conditioned by the physicochemical environment it works in at the same time. For instance, it is well-known that temperature affects the biochemical reactions rates. Therefore in free-living organisms that are able to grow at various environmental temperatures, such as the yeast Saccharomyces cerevisiae, evolution should have not only shaped the structural and functional properties of this key enzyme, but should have also provided mechanisms and pathways to adapt its activity to the optimal performance required. We studied the changes in RNA pol II elongation speed …

Cell volume homeostatically controls the rDNA repeat copy number and rRNA synthesis rate in yeast

[Abstract] The adjustment of transcription and translation rates to the changing needs of cells is of utmost importance for their fitness and survival. We have previously shown that the global transcription rate for RNA polymerase II in budding yeast Saccharomyces cerevisiae is regulated in relation to cell volume. Total mRNA concentration is constant with cell volume since global RNApol II-dependent nascent transcription rate (nTR) also keeps constant but mRNA stability increases with cell size. In this paper, we focus on the case of rRNA and RNA polymerase I. Contrarily to that found for RNA pol II, we detected that RNA polymerase I nTR increases proportionally to genome copies and cell s…

The mRNA degradation factor Xrn1 regulates transcription elongation in parallel to Ccr4

Abstract Co-transcriptional imprinting of mRNA by Rpb4 and Rpb7 subunits of RNA polymerase II (RNAPII) and by the Ccr4–Not complex conditions its post-transcriptional fate. In turn, mRNA degradation factors like Xrn1 are able to influence RNAPII-dependent transcription, making a feedback loop that contributes to mRNA homeostasis. In this work, we have used repressible yeast GAL genes to perform accurate measurements of transcription and mRNA degradation in a set of mutants. This genetic analysis uncovered a link from mRNA decay to transcription elongation. We combined this experimental approach with computational multi-agent modelling and tested different possibilities of Xrn1 and Ccr4 acti…

Xrn1 influence on gene transcription results from the combination of general effects on elongating RNA pol II and gene-specific chromatin configuration

mRNA homoeostasis is favoured by crosstalk between transcription and degradation machineries. Both the Ccr4-Not and the Xrn1-decaysome complexes have been described to influence transcription. While Ccr4-Not has been shown to directly stimulate transcription elongation, the information available on how Xrn1 influences transcription is scarce and contradictory. In this study we have addressed this issue by mapping RNA polymerase II (RNA pol II) at high resolution, using CRAC and BioGRO-seq techniques in Saccharomyces cerevisiae. We found significant effects of Xrn1 perturbation on RNA pol II profiles across the genome. RNA pol II profiles at 5ʹ exhibited significant alterations that were com…

Growth rate controls mRNA turnover in steady and non-steady states

Gene expression has been investigated in relation with growth rate in the yeast Saccharomyces cerevisiae, following different experimental strategies. The expression of some specific gene functional categories increases or decreases with growth rate. Our recently published results have unveiled that these changes in mRNA concentration with growth depend on the relative alteration of mRNA synthesis and decay, and that, in addition to this gene-specific transcriptomic signature of growth, global mRNA turnover increases with growth rate. We discuss here these results in relation with other previous and concurrent publications, and we add new evidence which indicates that growth rate controls m…

MOESM4 of Subtracting the sequence bias from partially digested MNase-seq data reveals a general contribution of TFIIS to nucleosome positioning

Additional file 4. Genes included in the different categories analyzed in this work.