0000000000076782
AUTHOR
Vicent Pelechano
showing 18 related works from this author
Rpb1 foot mutations demonstrate a major role of Rpb4 in mRNA stability during stress situations in yeast.
2016
The RPB1 mutants in the foot region of RNA polymerase II affect the assembly of the complex by altering the correct association of both the Rpb6 and the Rpb4/7 dimer. Assembly defects alter both transcriptional activity as well as the amount of enzyme associated with genes. Here, we show that the global transcriptional analysis of foot mutants reveals the activation of an environmental stress response (ESR), which occurs at a permissive temperature under optimal growth conditions. Our data indicate that the ESR that occurs in foot mutants depends mostly on a global post-transcriptional regulation mechanism which, in turn, depends on Rpb4-mRNA imprinting. Under optimal growth conditions, we …
The Lsm1-7/Pat1 complex binds to stress-activated mRNAs and modulates the response to hyperosmotic shock.
2018
RNA-binding proteins (RBPs) establish the cellular fate of a transcript, but an understanding of these processes has been limited by a lack of identified specific interactions between RNA and protein molecules. Using MS2 RNA tagging, we have purified proteins associated with individual mRNA species induced by osmotic stress, STL1 and GPD1. We found members of the Lsm1-7/Pat1 RBP complex to preferentially bind these mRNAs, relative to the non-stress induced mRNAs, HYP2 and ASH1. To assess the functional importance, we mutated components of the Lsm1-7/Pat1 RBP complex and analyzed the impact on expression of osmostress gene products. We observed a defect in global translation inhibition under…
The distribution of active RNA polymerase II along the transcribed region is gene-specific and controlled by elongation factors.
2010
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
2012
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…
Genomic-Wide Methods to Evaluate Transcription Rates in Yeast
2011
Gene transcription is a dynamic process in which the desired amount of an mRNA is obtained by the equilibrium between its transcription (TR) and degradation (DR) rates. The control mechanism at the RNA polymerase level primarily causes changes in TR. Despite their importance, TRs have been rarely measured. In the yeast Saccharomyces cerevisiae, we have implemented two techniques to evaluate TRs: run-on and chromatin immunoprecipitation of RNA polymerase II. These techniques allow the discrimination of the relative importance of TR and DR in gene regulation for the first time in a eukaryote.
A genomic view of mRNA turnover in yeast
2011
The steady-state mRNA level is the result of two opposing processes: transcription and degradation; both of which can provide important points to regulate gene expression. In the model organism yeast Saccharomyces cerevisiae, it is now possible to determine, at the genomic level, the transcription and degradation rates, as well as the mRNA amount, using DNA chip or parallel sequencing technologies. In this way, the contribution of both rates to individual and global gene expressions can be analysed. Here we review the techniques used for the genomic evaluation of the transcription and degradation rates developed for this yeast, and we discuss the integration of the data obtained to fully an…
Regulon-Specific Control of Transcription Elongation across the Yeast Genome
2009
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…
A genomic study of the inter-ORF distances in Saccharomyces cerevisiae.
2006
The genome of eukaryotic microbes is usually quite compacted. The yeast Saccharomyces cerevisiae is one of the best-known examples. Open reading frames (ORFs) occupy about 75% of the total DNA sequence. The existence of other, non-protein coding genes and other genetic elements leaves very little space for gene promoters and terminators. We have performed an in silico study of inter-ORF distances that shows that there is a minimum distance between two adjacent ORFs that depends on the relative orientation between them. Our analyses suggest that different kinds of promoters and terminators exist with regard to their length and ability to overlap each other. The experimental testing of some p…
eIF5A facilitates translation termination globally and promotes the elongation of many non polyproline-specific tripeptide sequences
2017
Abstract eIF5A is an essential protein involved in protein synthesis, cell proliferation and animal development. High eIF5A expression is observed in many tumor types and has been linked to cancer metastasis. Recent studies have shown that eIF5A facilitates the translation elongation of stretches of consecutive prolines. Activated eIF5A binds to the empty E-site of stalled ribosomes, where it is thought to interact with the peptidyl-tRNA situated at the P-site. Here, we report a genome-wide analysis of ribosome stalling in Saccharomyces cerevisiae eIF5A depleted cells using 5Pseq. We confirm that, in the absence of eIF5A, ribosomes stall at proline stretches, and extend previous studies by …
Dynamic remodeling of histone modifications in response to osmotic stress in Saccharomyces cerevisiae.
2014
Abstract Background Specific histone modifications play important roles in chromatin functions; i.e., activation or repression of gene transcription. This participation must occur as a dynamic process. Nevertheless, most of the histone modification maps reported to date provide only static pictures that link certain modifications with active or silenced states. This study, however, focuses on the global histone modification variation that occurs in response to the transcriptional reprogramming produced by a physiological perturbation in yeast. Results We did a genome-wide chromatin immunoprecipitation analysis for eight specific histone modifications before and after saline stress. The most…
The ribosome assembly gene network is controlled by the feedback regulation of transcription elongation
2017
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…
The transcriptional inhibitor thiolutin blocks mRNA degradation in yeast.
2007
Thiolutin is commonly used as a general inhibitor of transcription in yeast. It has been used to calculate mRNA decay rates by stopping the transcription and then determining the relative abundance of individual mRNAs at different times after inhibition. We report here that thiolutin is also an inhibitor of mRNA degradation, and thus its use can lead to miscalculations of mRNA half-lives. The inhibition of mRNA decay seems to affect the mRNA degradation pathway without impeding poly(A) shortening, given that the decay rate of total poly(A) amount is not reduced by thiolutin. Moreover, the thiolutin-dependent inhibition of mRNA degradation has variable effects on different functional groups …
A complete set of nascent transcription rates for yeast genes
2010
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 cellular growth rate controls overall mRNA turnover, and modulates either transcription or degradation rates of particular gene regulons
2015
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…
There is a steady-state transcriptome in exponentially growing yeast cells
2010
The growth of yeast cells in batches in glucose-based media is a standard condition in most yeast laboratories. Most gene expression experiments are done by taking this condition as a reference. Presumably, cells are in a stable physiological condition that can be easily reproduced in other laboratories. With this assumption, however, it is necessary to consider that the average amount of the mRNAs per cell for most genes does not change during exponential growth. That is to say, there is a steady-state condition for the transcriptome. However, this has not been rigorously demonstrated to date. In this work we take several cell samples during the exponential phase growth to perform a kineti…
Biotin-Genomic Run-On (Bio-GRO): A High-Resolution Method for the Analysis of Nascent Transcription in Yeast
2016
Transcription is a highly complex biological process, with extensive layers of regulation, some of which remain to be fully unveiled and understood. To be able to discern the particular contributions of the several transcription steps it is crucial to understand RNA polymerase dynamics and regulation throughout the transcription cycle. Here we describe a new nonradioactive run-on based method that maps elongating RNA polymerases along the genome. In contrast with alternative methodologies for the measurement of nascent transcription, the BioGRO method is designed to minimize technical noise that arises from two of the most common sources that affect this type of strategies: contamination wi…
Chromatin-dependent regulation of RNA polymerases II and III activity throughout the transcription cycle
2015
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…
Rpb4 and Puf3 imprint and post-transcriptionally control the stability of a common set of mRNAs in yeast
2020
ABSTRACTGene expression involving RNA polymerase II is regulated by the concerted interplay between mRNA synthesis and degradation, crosstalk in which mRNA decay machinery and transcription machinery respectively impact transcription and mRNA stability. Rpb4, and likely dimer Rpb4/7, seem the central components of the RNA pol II governing these processes. In this work we unravel the molecular mechanisms participated by Rpb4 that mediate the posttranscriptional events regulating mRNA imprinting and stability. By RIP-Seq, we analyzed genome-wide the association of Rpb4 with mRNAs and demonstrated that it targeted a large population of more than 1400 transcripts. A group of these mRNAs was als…