Análisis del papel del pie de la RNA polimerasa II de Saccharomyces cerevisiae y su importancia en múltiples etapas de la transcripción

[ES] Se conoce poco sobre los mecanismos que gobiernan el ensamblaje nuclear, estabilidad, degradación y reciclaje de la RNA polimerasa II. Demostramos que el pie de la RNA pol II es crucial para su ensamblaje y estabilidad, asegurando la correcta asociación de Rpb I-Rpb6 Y del dímero Rpb4/7. Mutaciones en el pie afectan al ensamblaje y estabilidad de la enzima, lo que es suprimido por la sobreexpresión de RPB6, provocan degradación nuclear de Rpbl dependiente de Rsp5 y conducen a alteraciones de diversas etapas transcripcionales. El defecto de ensamblaje altera la actividad transcripcional, la cantidad de enzima asociada a los genes, la fosforilación del CTD, la maquinaria de capping del mRNA, sugiere un incremento en la RNA pol II parada y altera la estabilidad del PIe. Los mutantes del pie muestran una respuesta a estrés así como una reducción en la cantidad de mRNA unidos a Rpb4/7 que altera su degradación[EN] Little is known about the mechanisms governing the nuclear assembly, stability, degradation, and recycling of RNA polymerase II. We demonstrate that the foot ofthe RNA pol II is crucial for the assembly and stability ofthe complex, by ensuring the correct association of Rpb l-Rpb6, and of the dimer Rpb4/7. Mutations at the foot affect the assembly and stability ofthe enzyme, a defect that is offset by RPB6 overexpression, cause Rpbl degradation in the nuc\eus by an Rsp5-dependent mechanism, and leads to alterations in several transcription steps. Assembly defect alter transcriptional activity, the amount of enzyme associated with the genes, the CTD phosphorylation, interferes with mRNA capping machinery, suggest an increase in stalled RNA pol II and alter the PIC stability. Foot mutants show a stress response and a reduction in the amount of mRNA bound to Rpb4/7, affecting the mRNA decay.Tesis Univ. Jaén. Departamento de Biología Experimental, leída el 18 de marzo de 201

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

Phytosociological characteristic of Latvian beaches and dunes within the context of the Baltic Sea region

Anotācija Laime B. 2010. Latvijas kāpu un pludmaļu fitosocioloģiskais raksturojums Baltijas jūras reģiona kontekstā Pētījumā raksturota piekrastes veģetācija 5998 parauglaukumos. Izmantojot TWINSPAN, DCA metodi un JUICE programmu, izstrādāta Latvijas piekrastes veģetācijas sintaksonomiskā klasifikācija, kas ietver 5 klases un 9 asociācijas: Juncetum bufonii, Atriplicetum littoralis, Cakiletum maritimae, Honckenyetum peploidis, Elymo-Ammophiletum arenariae, Festucetum polesicae, Caricetum arenariae, Corniculario aculatae-Corynephoretum canescentis un Carici-Callunetum. Raksturota katras asociācijas veģetācija, ekoloģija, izplatība un aizsardzība. Nodalīti vairāki sukcesiju tipi. Iegūtie rezultāti salīdzināti ar līdzīgiem pētījumiem Baltijas jūras reģionā. Atslēgvārdi: veģetācija, augu sabiedrība, Ammophiletea, Cakiletea, Koelerio-CorynephoreteaAnnotation Laime B. 2010. Phytosociological characteristic of Latvian beaches and dunes within the context of the Baltic Sea region Vegetation in 5998 sample plots was described. Using TWINSPAN, DCA method and program JUICE, the syntaxonomical classification of Latvian coastal vegetation was carried out, distinguished 5 classes, 9 associations: Juncetum bufonii, Atriplicetum littoralis, Cakiletum maritimae, Honckenyetum peploidis, Elymo-Ammophiletum arenariae, Festucetum polesicae, Caricetum arenariae, Corniculario aculatae-Corynephoretum canescentis and Carici-Callunetum. Vegetation, ecology, distribution and protection of each syntaxon, and several types of succession were characterised. Results are compared with similar communities in the Baltic Sea region. Key words: vegetation, plant communities, Ammophiletea, Cakiletea, Koelerio-Corynephorete

Iwr1 nuclear localization is independent of Bud27.

<p>Live cell imaging of Iwr1-Gfp deleted for its NES domain in wild-type and Δ<i>bud27</i> cells at 30°C.</p

Lack of Bud27 led to RNA pol I, II, and III cytoplasmic accumulation.

<p>A) Immunocytochemistry experiments using antibodies against Rpa190-HA (anti-HA), Rpb1 (8WG16), and Rpc160-Myc (anti-Myc) in wild-type and Δ<i>bud27</i> mutant cells with tagged Rpa190-HA (RNA pol I) and Rpc160-Myc (RNA pol III), at 30°C. B) Live cell imaging of Rpb8-ECFP in wild-type and Δ<i>bud27</i> mutant cells, at 30°C. C) Western blot of chromatin fractions from wild-type and Δ<i>bud27</i> mutant cells harbouring an empty vector (pCM) or a vector overexpressing <i>BUD27</i> (<i>pCM-BUD27-TAP</i>). Tubulin and Nop1 were used as controls of non-chromatin and chromatin fractions, respectively. D) Rpb1 immunolocalisation analysis (8WG16) in human pulmonary fibroblast under silencing of URI. As a control, cells without siRNA heteroduplex.</p

Δ<i>bud27 mutant</i> phenotypes are corrected by overexpression of different <i>BUD27</i> constructions and <i>RPB5</i>.

<p>A) Growth of wild-type and Δ<i>bud27</i> mutant strains transformed with vectors overexpressing <i>RPB5</i>, at different temperatures or in the presence of rapamycin. <i>pCM</i> and <i>pFL</i> correspond to the control empty vectors. B) Live cell imaging of Rpb8-ECFP in wild-type and Δ<i>bud27</i> mutant cells at 30°C, containing empty vector (<i>pFL</i>) or overexpressing <i>RPB5</i> (<i>pFL-RPB5</i>).</p

Bud27 physically interacts with the three RNA polymerases.

<p>A) Growth of BUD27-TAP, Wild-type and a Δ<i>bud27</i> cells inYPD at the indicated temperatures. B) Summary of the proteins interacting with Bud27 in a TAP purification analysis. C) Western blot of protein co-purified with Bud27-TAP (3), Rpb3-TAP (2), and non- tagged wild-type strains (1). anti-HA, anti-Myc, anti-Rpb1 (8WG16), and anti-PAP antibodies were used. No tag: wild-type strain BY4741; Rpb3-TAP: Rpb3-TAP, Rpa190-HA (RNA pol I) and Rpc25-Myc (RNA pol III) strain; Bud27-TAP: Bud27-TAP, Rpa190-HA (RNA pol I) and Rpc25-Myc (RNA pol III) strain.</p

RNA pol I, II, and III nuclear localization is independent of the chaperone prefoldin (PFD) complex.

<p>Live cell imaging of Rpb8-ECFP and Rpb1-GFP in Δ<i>bud27</i>, Δ<i>gim6</i> (Δ<i>pfd1</i>), Δ<i>gim4</i> (Δ<i>pfd2</i>), and Δ<i>yke2</i> (Δ<i>pfd6</i>) mutant cells containing C-terminal ECFP tagged Rpb8 and C-terminal GFP tagged Rpb1, at 30°C.</p