The role of RNA Polymerase II-dependent transcription elongation in the cross-talk between mRNA synthesis and decay.

The main molecule in gene expression is messenger RNA (mRNA) which transfers the information contained in genes in the nucleus to the cytoplasm where it is translated into proteins that carry out cellular functions. mRNA levels are determined through its synthesis, by the RNA polymerase II, and degradation, which involves the Ccr4-Not complex and Xrn1. It has become increasingly apparent that the mRNA concentration in a cell is maintained at a particular level even through stressful situations. The way the cell is able to do this is by a cross-talk between the machinery responsible for its transcription and that responsible for its degradation. In this work we have attempted to unravel the mechanisms by which this cross-talk occurs. For this complex task, we first studied how transcription and degradation was affected after deleting a single gene known to be involved in either one of these mechanisms. This study confirmed the existence of a strong feedback between mRNA synthesis and decay, and also helped us uncover some of the elements important for this cross-talk. The most interesting finding was the correlation between transcription elongation and mRNA degradation, suggesting that it is directly relevant for cross-talk. Second, we mathematically modelled and computationally simulated this coupling between transcription and mRNA decay. Thanks to in silico experimentation, we found that two proteins involved in degradation (Ccr4-Not and Xrn1) were most likely also involved in transcription, and therefore the feedback mechanism. This result complements that of the first study and places both Ccr4-Not and Xrn1 as important proteins for cross-talk. Finally, we analysed the exonuclease Xrn1 in depth through genome-wide experiments. This study allowed us to conclude that Xrn1 is directly involved in transcription and influence both early and late RNA polymerase II-dependent transcription elongation. The results of this thesis have enabled us to come up with a model for how the cross-talk could work in yeast cells and allowed us to envision new hypotheses to explain the novel results.Premio Extraordinario de Doctorado U

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

Co-transcriptional imprinting of mRNA by Rpb4 and Rpb7 subunits of RNA polymerase II (RNAPII) and by the Ccr4–Not complex conditions its posttranscriptional 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 action in gene transcription. This double strategy brought us to conclude that both Xrn1-decaysome and Ccr4–Not regulate RNAPII elongation, and that they do it in parallel. We validated this conclusion measuring TFIIS genome-wide recruitment to elongating RNAPII. We found that xrn1Δ and ccr4Δ exhibited very different patterns of TFIIS versus RNAPII occupancy, which confirmed their distinct role in controlling transcription elongation. We also found that the relative influence of Xrn1 and Ccr4 is different in the genes encoding ribosomal proteins as compared to the rest of the genome

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 conditions, Spt6 was physically targeted to the upregulated genes, where it helped maintain their chromatin integrity and the synthesis of properly stable mRNAs. The mRNA profiles of a large set of ribosome biogenesismutants confirmed the existence of a feedback regulatory network among ribosome assembly genes. The transcriptional response in this network depended on both the specific malfunction and the role of the regulated gene. In accordance with our screening, Spt6 positively contributed to the optimal operation of this global network. On the whole, this work uncovers a feedback control of ribosome biogenesis by fine-tuning transcription elongation in ribosome assembly factor-coding genes.Ministerio de Economía y Competitividad BFU2013-48643-C3-1-P, BFU2016-77728-C3-1-P, BFU2013-48643-C3- 3-P, BFU2013-42958-PJunta de Andalucía P12-BIO1938MO, P08-CVI-03508Comunidad Valenciana 2015/00

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 actual mRNA synthesis rate constant. This cannot apply to the yeast Saccharomyces cerevisiae, where this mechanism would provoke a never-ending increasing mRNA synthesis rate in smaller daughter cells. We show here that, contrarily to other eukaryotes with symmetric division, budding yeast keeps the nascent transcription rates of its RNA polymerases constant and increases mRNA stability. This control on RNA pol II-dependent transcription rate is obtained by controlling the cellular concentration of this enzyme

Identifying and understanding factors that affect the translation of therapies from the laboratory to patients: a study protocol

Background: The process of translating preclinical findings into a clinical setting takes decades. Previous studies have suggested that only 5-10% of the most promising preclinical studies are successfully translated into viable clinical applications. The underlying determinants of this low success rate (e.g. poor experimental design, suboptimal animal models, poor reporting) have not been examined in an empirical manner. Our study aims to determine the contemporary success rate of preclinical-to-clinical translation, and subsequently determine if an association between preclinical study design and translational success/failure exists. Methods: Established systematic review methodology will be used with regards to the literature search, article screening and study selection process. Preclinical, basic science studies published in high impact basic science journals between 1995 and 2015 will be included. Included studies will focus on publicly available interventions with potential clinical promise. The primary outcome will be successful clinical translation of promising therapies - defined as the conduct of at least one Phase II trial (or greater) with a positive finding. A case-control study will then be performed to evaluate the association between elements of preclinical study design and reporting and the likelihood of successful translation. Discussion: This study will provide a comprehensive analysis of the therapeutic translation from the laboratory bench to the bedside. Importantly, any association between factors of study design and the success of translation will be identified. These findings may inform future research teams attempting preclinical-to-clinical translation. Results will be disseminated to identified knowledge users that fund/support preclinical research

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 compatible with decreased elongation rates in the absence of Xrn1. Nucleosome mapping detected altered chromatin configuration in the gene bodies. We also detected accumulation of RNA pol II shortly upstream of polyadenylation sites by CRAC, although not by BioGRO-seq, suggesting higher frequency of backtracking before pre-mRNA cleavage. This phenomenon was particularly linked to genes with poorly positioned nucleosomes at this position. Accumulation of RNA pol II at 3ʹ was also detected in other mRNA decay mutants. According to these and other pieces of evidence, Xrn1 seems to influence transcription elongation at least in two ways: by directly favouring elongation rates and by a more general mechanism that connects mRNA decay to late elongation.Ministerio de Economía y Competitividad BFU2016-77728- C3-1-P, BFU2016-77728-C3-3-P, BFU2016- 77728-C3-2-P, RED2018-102467-TJunta de Andalucía BIO271, US-1256285, BIO258, UJA 1260360Generalitat Valenciana AICO/2019/08

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 compatible with decreased elongation rates in the absence of Xrn1. Nucleosome mapping detected altered chromatin configuration in the gene bodies. We also detected accumulation of RNA pol II shortly upstream of polyadenylation sites by CRAC, although not by BioGRO-seq, suggesting higher frequency of backtracking before pre-mRNA cleavage. This phenomenon was particularly linked to genes with poorly positioned nucleosomes at this position. Accumulation of RNA pol II at 3ʹ was also detected in other mRNA decay mutants. According to these and other pieces of evidence, Xrn1 seems to influence transcription elongation at least in two ways: by directly favouring elongation rates and by a more general mechanism that connects mRNA decay to late elongation.This work has been supported by grants from the Ministerio de Economía, Industria y Competitividad – Agencia Estatal de Investigación, and European Union funds (FEDER) [BFU2016-77728-C3-1-P to S. C.],[BFU2016-77728-C3-3-P to J.E.P-O & P.A], [BFU2016-77728-C3-2-P to F.N.] and RED2018‐102467‐T to J.E.P-O, F.N. and S.C.; by FPI grants from the Spanish Government to V.B and A.C-B; by the Regional Andalusian Government [BIO271 and US-1256285 to S. C.], [BIO258 and FEDER-UJA 1260360 to F.N.] and from the Regional Valencian Government [AICO/2019/088 to J.E.P-O]. Funding for open access charge: [BFU2016-77728-C3-1-P]

Using perceived impacts, governance and social indicators to explain support for protected areas

Protected areas (PAs) are the foremost policy tool for biodiversity conservation internationally. In order for PAs to deliver desired conservation outcomes effectively, equitably, and for the long-term, they require a high level of support from local communities. A mosaic of factors have been proposed aiming to explain the level of support for PAs focusing mainly on governance indicators, perceived social impacts and social attributes, such as personal norms and values. However, these factors are often explored in isolation and in studies of small scale within the scientific literature. To cover this research gap we run a structural equation model exploring how the interconnections between these factors can lead to higher levels of support for PAs using data from 3239 local residents in 10 PAs in Europe. Our analysis shows that the mediation effects of governance indicators, such as trust in institutions and level of public engagement, are important in explaining associations between an individual's social profile and their perceived social impacts with public support. Our findings also provide a useful and operational framework for PA practitioners and researchers illuminating pathways to increase the level of public support for a PA

Nursing sensitive outcomes in patients with rheumatoid arthritis: A systematic literature review

© 2017 Elsevier Ltd Background Although rheumatology nursing has been shown to be effective in managing patients with rheumatoid arthritis, patient outcomes sensitive to nursing interventions (nursing sensitive outcomes) have not been systematically studied. Objectives The objective of this study was to identify and delineate relevant patient outcomes measured in studies that reported nursing interventions in patients with rheumatoid arthritis. Design A systematic search was conducted from 1990 to 2016. Inclusion criteria were (i) patients with rheumatoid arthritis, (ii) adult population age ≥16 years, (iii) nurse as part of the care team or intervention delivery, (iv) primary research only, (v) English language, and (vi) quantitative studies with nursing sensitive outcomes. Data sources Medline, CINAHL, Ovid nursing, Cochrane library and PsycINFO databases were searched for relevant studies. Review methods Using the predetermined inclusion/exclusion criteria, nine reviewers working in pairs assessed the eligibility of the identified studies based on titles and abstracts. Papers meeting the inclusion criteria were retrieved and full texts were further assessed. Critical Appraisal Skills Programme tools were used to assess the quality of the included studies. Data on nursing sensitive outcomes were extracted independently by two reviewers. The Outcome Measures in Rheumatology comprehensive conceptual framework for health was used to contextualise and present findings. Results Of the 820 articles retrieved, 7 randomised controlled trials and 3 observational studies met the inclusion criteria. Seventeen nursing sensitive outcomes were identified (disease activity, clinical effects, pain, early morning stiffness duration, fatigue, patient safety issues, function, knowledge, patient satisfaction, confidence in care received, mental health status, self-efficacy, patient attitude/perception of ability to control arthritis, quality of life, health utility, health care resources, death). These fitted into 10 health intervention domains in keeping with the pre-specified conceptual framework for health: disease status, effectiveness, safety, function, knowledge, satisfaction, psychological status, quality of life, cost, death. A total of 59 measurement instruments were identified comprising patient reported outcome measures (n = 31), and biologic measures and reports (n = 28). Conclusions This review is notable in that it is the first to have identified, and reported, a set of multidimensional outcome measures that are sensitive to nursing interventions in rheumatology specifically. Further research is required to determine a core set of outcomes to be used in all rheumatology nursing intervention studies