INFLUENCIA DE Bud27 Y Rtr1 EN EL CICLO DE VIDA DE LOS RNA

Bud27 (prefolin-like) y Rtr1 (fosfatasa del CTD de la RNA polimerasa II) en Saccharomyces cerevisiae, son dos proteínas implicadas en el ensamblaje y/o transporte de las RNA polimerasas al núcleo. En este trabajo se ha estudiado su influencia en el ciclo de vida de los mRNA. Para ello se ha desarrollado un método de purificación de fracciones cromatínicas enriquecidas con sus proteínas asociadas denominado yChEFs (yeast Chromatin Enriched Fractions), con el que hemos determinado el proteoma asociado a cromatina así como el proteoma asociado a cromatina dependiente de RNA. El estudio del papel de Bud27 en el ciclo de vida del mRNA, mostró que la falta de Bud27 conlleva una disminución en la estabilidad de los mRNA y una alteración en la selección de los sitios de poliadenilación. Rtr1 por su parte modula negativamente la estabilidad de los transcritos en un mecanismo putativamente coordinado con las proteínas Rpb4 y Dhh1.Bud27 (prefoldin-like) and Rtr1 (CTD phosphatase of the RNA polymerase II) in Saccharomyces cerevisiae, are two proteins involved in the assembly and/or transport of RNA pol to the nucleus. In this work we have studied their influence on the mRNA life cycle. For that, we have developed a method to purify chromatin enriched fractions with their associated proteins named yChEFs (yeast Chromatin Enriched Fractions). The procedure has been used to analyze the chromatin-associated proteome and the RNA-dependent chromatin-associated proteome. Studies of the role of Bud27 in mRNA life cycle show that the lack of Bud27 leads to an overall decrease in mRNA stability and to an alteration in the polyadenylation sites recognition. In other hand, Rtr1 plays a role in the mRNA life cycle, modulating negatively their stability, probably in a coordinated mechanism with Rpb4 and Dhh1.Tesis Univ. Jaén. Departamento de Biología Experimental. Leída el 9 de abril de 2018

Subunits Common to RNA Polymerases

RNA polymerases are heteromultimeric complexes responsible of RNA synthesis. In yeast, as in the other eukaryotes, these complexes contain five common subunits (Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12) that must have similar functions in the three RNA polymerases. However, some of these proteins have been shown to also have specific roles. In the last few decades, substantial progress has been made to understand the role of these common subunits in transcription, but their participation in the activity of each enzyme remains unclear. This review gives a comprehensive overview of current knowledge on the five common subunits of eukaryotic RNA pol, placing attention not only on their common roles in the activity of the RNA pols but also on describing specific roles for some of the complexes

Non spontaneous saccadic movements identification in clinical electrooculography using machine learning

In this paper we evaluate the use of the machine learning algorithms Support Vector Machines, K-Nearest Neighbors, CART decision trees and Naive Bayes to identify non spontaneous saccades in clinical electrooculography tests. Our approach tries to solve problems like the use of manually established thresholds present in classical methods like identification by velocity threshold (I-VT) or identification by dispersion threshold (I-DT). We propose a modification to an adaptive threshold estimation algorithm for detecting signal impulses without the need of any user input. Also, a set of features were selected to take advantage of intrinsic characteristics of clinical electrooculography tests. The models were evaluated with signals recorded to subjects affected by Spinocerebellar Ataxia type 2 (SCA2). Results obtained by the algorithm shows accuracies over 97%, recalls over 97% and precisions over 91% for the four models evaluated.Universidad de Málaga, Campus de excelencia de Andalucía Tec

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 yeast prefoldin-like URI-orthologue Bud27 associates with the RSC nucleosome remodeler and modulates transcription

Bud27, the yeast orthologue of human URI/RMP, is a member of the prefoldin-like family of ATPindependent molecular chaperones. It has recently been shown to mediate the assembly of the three RNA polymerases in an Rpb5-dependent manner. In this work, we present evidence of Bud27 modulating RNA pol II transcription elongation. We show that Bud27 associates with RNA pol II phosphorylated forms (CTD-Ser5P and CTD-Ser2P), and that its absence affects RNA pol II occupancy of transcribed genes. We also reveal that Bud27 associatesin vivo with the Sth1 component of the chromatin remodeling complex RSC and mediates its association with RNA pol II. Our data suggest that Bud27, in addition of contributing to Rpb5 folding within the RNA polymerases, also participates in the correct assembly of other chromatin-associated protein complexes, such as RSC, thereby modulating their activit

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]

The Association of Rpb4 with RNA Polymerase II Depends on CTD Ser5P Phosphatase Rtr1 and Influences mRNA Decay in <i>Saccharomyces cerevisiae</i>

Rtr1 is an RNA polymerase II (RNA pol II) CTD-phosphatase that influences gene expression during the transition from transcription initiation to elongation and during transcription termination. Rtr1 interacts with the RNA pol II and this interaction depends on the phosphorylation state of the CTD of Rpb1, which may influence dissociation of the heterodimer Rpb4/7 during transcription. In addition, Rtr1 was proposed as an RNA pol II import factor in RNA pol II biogenesis and participates in mRNA decay by autoregulating the turnover of its own mRNA. Our work shows that Rtr1 acts in RNA pol II assembly by mediating the Rpb4/7 association with the rest of the enzyme. RTR1 deletion alters RNA pol II assembly and increases the amount of RNA pol II associated with the chromatin that lacks Rpb4, decreasing Rpb4-mRNA imprinting and, consequently, increasing mRNA stability. Thus, Rtr1 interplays RNA pol II biogenesis and mRNA decay regulation. Our data also indicate that Rtr1 mediates mRNA decay regulation more broadly than previously proposed by cooperating with Rpb4. Interestingly, our data include new layers in the mechanisms of gene regulation and in the crosstalk between mRNA synthesis and decay by demonstrating how the association of Rpb4/7 to the RNA pol II influences mRNA decay

Prefoldin-like Bud27 influences the transcription of ribosomal components and ribosome biogenesis in Saccharomyces cerevisiae

Understanding the functional connection that occurs for the three nuclear RNA polymerases to synthesize ribosome components during the ribosome biogenesis process has been the focal point of extensive research. To preserve correct homeostasis on the production of ribosomal components, cells might require the existence of proteins that target a common subunit of these RNA polymerases to impact their respective activities. This work describes how the yeast prefoldin-like Bud27 protein, which physically interacts with the Rpb5 common subunit of the three RNA polymerases, is able to modulate the transcription mediated by the RNA polymerase I, likely by influencing transcription elongation, the transcription of the RNA polymerase III, and the processing of ribosomal RNA. Bud27 also regulates both RNA polymerase II-dependent transcription of ribosomal proteins and ribosome biogenesis regulon genes, likely by occupying their DNA ORFs, and the processing of the corresponding mRNAs. With RNA polymerase II, this association occurs in a transcription rate-dependent manner. Our data also indicate that Bud27 inactivation alters the phosphorylation kinetics of ribosomal protein S6, a readout of TORC1 activity. We conclude that Bud27 impacts the homeostasis of the ribosome biogenesis process by regulating the activity of the three RNA polymerases and, in this way, the synthesis of ribosomal components. This quite likely occurs through a functional connection of Bud27 with the TOR signaling pathway