Estudio funcional de factores implicados en la transcripción y exportación de los RNAs en Saccharomyces cerevisiae

La expresión génica en eucariotas ocurre a través del acoplamiento de diferentes procesos finamente regulados e interconectados en los que participan varios complejos multiprotéicos. Nuestro trabajo se centra en el estudio de las conexiones entre esos procesos que aseguran una correcta formación del ARN. La proteína Sus1 es un factor esencial para el correcto acoplamiento entre la transcripción y la exportación de los ARN mensajeros para su traducción en el citoplasma (1, 2). Sus1 es componente de 3 complejos diferentes: -TREX-2, asociado al complejo del poro nuclear y tiene funciones en el metabolismo del ARN mensajero, la exportación del mismo al citoplasma y en estabilidad genómica; -SAGA, un co-activador transcripcional con actividades de acetilación de la histona H3 y desubicuitinación de la histona H2B. De esta forma SAGA está directamente implicado en la regulación de la expresión de aproximadamente el 10% del genoma de la levadura, sobretodo de los genes que se inducen bajo situaciones de estrés celular; - y por último SLIK, un complejo similar a SAGA cuyas únicas diferencias con éste son la ausencia del factor Spt8 y la pérdida del extremo C-terminal del factor Spt7 (3). Aunque el complejo SAGA y sus funciones están ampliamente estudiadas en levaduras y eucariotas superiores, las funciones concretas del complejo SLIK todavía siguen siendo en gran parte desconocidas. Estudios sobre la organización de los diferentes componentes de complejos multiprotéicos demostraron que las subunidades de SAGA y SLIK se ensamblan formando 4 submódulos diferentes (4). El denominado módulo DUB está formado por 4 subunidades entre las cuales se encuentran Sus1 y la proteasa Ubp8 encargada de la deubicuitilación de H2B. A pesar de que durante años se planteaba necesaria la presencia e interacción de los 4 submódulos de SAGA/SLIK para llevar acabo sus funciones, recientemente y en concordancia con resultados tanto nuestros como de otros laboratorios, se ha propuesto la posibilidad de que el submódulo DUB sea capaz de ser activo y funcional en un contexto diferente a SAGA/SLIK (5) y relacionado con funciones reguladoras de la transcripción. Con el fin de elucidar y profundizar en las funciones de Sus1 como componente de los complejos SAGA, SLIK y TREX-2, así como a través de sus relaciones con otros factores implicados en la expresión génica, establecimos tres objetivos. Primero, estudiar las funciones del complejo SLIK a través de Sus1 tanto en la transcripción como en la exportación de los RNAs. Segundo, profundizar en las funciones del módulo de desubicuitinación (DUB) de los complejos SAGA/SLIK, así como sus mecanismos de regulación y el acoplamiento de sus funciones con la exportación de los mRNAs. Y tercero estudiar la relación física y funcional de Sus1 con otros factores de transcripción y proteínas relacionadas con la expresión génica. 1.- García-Oliver E, García-Molinero V, Rodríguez-Navarro S. mRNA export and gene expression: the SAGA-TREX-2 connection. Biochim Biophys Acta. 2012 Jun. 2.- Galán A, Rodríguez-Navarro S. Sus1/ENY2: a multitasking protein in eukaryotic gene expression. Crit Rev Biochem Mol Biol. 2012 Nov-Dec. 3.- Spedale G, Mischerikow N, Heck AJ, Timmers HT, Pijnappel WW. Identification of Pep4p as the protease responsible for formation of the SAGA-related SLIK protein complex. J Biol Chem. 2010 Jul. 4.- Lee KK, Sardiu ME, Swanson SK, Gilmore JM, Torok M, Grant PA, Florens L, Workman JL, Washburn MP. Combinatorial depletion analysis to assemble the network architecture of the SAGA and ADA chromatin remodeling complexes. Mol Syst Biol. 2011 Jul. 5.- Lim S, Kwak J, Kim M, Lee D. Separation of a functional deubiquitylating module from the SAGA complex by the proteasome regulatory particle. Nat Commun. 2013

A novel link between Sus1 and the cytoplasmic mRNA decay machinery suggests a broad role in mRNA metabolism

9 páginas, 5 figuras. : Supplementary tables. Yeast strains and plasmids used in this study: http://www.biomedcentral.com/content/supplementary/1471-2121-11-19-S1.PDFBACKGROUND: Gene expression is achieved by the coordinated action of multiple factors to ensure a perfect synchrony from chromatin epigenetic regulation through to mRNA export. Sus1 is a conserved mRNA export/transcription factor and is a key player in coupling transcription initiation, elongation and mRNA export. In the nucleus, Sus1 is associated to the transcriptional co-activator SAGA and to the NPC associated complex termed TREX2/THSC. Through these associations, Sus1 mediates the nuclear dynamics of different gene loci and facilitate the export of the new transcripts. RESULTS: In this study, we have investigated whether the yeast Sus1 protein is linked to factors involved in mRNA degradation pathways. We provide evidence for genetic interactions between SUS1 and genes coding for components of P-bodies such as PAT1, LSM1, LSM6 and DHH1. We demonstrate that SUS1 deletion is synthetic lethal with 5'-->3' decay machinery components LSM1 and PAT1 and has a strong genetic interaction with LSM6 and DHH1. Interestingly, Sus1 overexpression led to an accumulation of Sus1 in cytoplasmic granules, which can co-localise with components of P-bodies and stress granules. In addition, we have identified novel physical interactions between Sus1 and factors associated to P-bodies/stress granules. Finally, absence of LSM1 and PAT1 slightly promotes the Sus1-TREX2 association. CONCLUSIONS: In this study, we found genetic and biochemical association between Sus1 and components responsible for cytoplasmic mRNA metabolism. Moreover, Sus1 accumulates in discrete cytoplasmic granules, which partially co-localise with P-bodies and stress granules under specific conditions. These interactions suggest a role for Sus1 in gene expression during cytoplasmic mRNA metabolism in addition to its nuclear function.SRN is supported by the Ramón y Cajal program and funded by the Spanish Ministry of Science and Innovation (BFU2008-04082-C02-02) and Generalitat Valenciana (ACOMP2009/053). BCB is a holder of a predoctoral fellowship from the Spanish Ministry of Science and Innovation.Peer reviewe

SUS1 introns are required for efficient mRNA nuclear export in yeast

Efficient coupling between mRNA synthesis and export is essential for gene expression. Sus1/ENY2, a component of the SAGA and TREX-2 complexes, is involved in both transcription and mRNA export. While most yeast genes lack introns, we previously reported that yeast SUS1 bears two. Here we show that this feature is evolutionarily conserved and critical for Sus1 function. We determine that while SUS1 splicing is inefficient, it responds to cellular conditions, and intronic mutations either promoting or blocking splicing lead to defects in mRNA export and cell growth. Consistent with this, we find that an intron-less SUS1 only partially rescues sus1Δ phenotypes. Remarkably, splicing of each SUS1 intron is also affected by the presence of the other and by SUS1 exonic sequences. Moreover, by following SUS1 RNA and protein levels we establish that nonsense-mediated decay (NMD) pathway and the splicing factor Mud2 both play a role in SUS1 expression. Our data (and those of the accompanying work by Hossain et al.) provide evidence of the involvement of splicing, translation, and decay in the regulation of early events in mRNP biogenesis; and imply the additional requirement for a balance in splicing isoforms from a single gene

Fine Chromatin-Driven Mechanism of Transcription Interference by Antisense Noncoding Transcription

Eukaryotic genomes are almost entirely transcribed by RNA polymerase II. Consequently, the transcription of long noncoding RNAs often overlaps with coding gene promoters, triggering potential gene repression through a poorly characterized mechanism of transcription interference. Here, we propose a comprehensive model of chromatin-based transcription interference in Saccharomyces cerevisiae (S. cerevisiae). By using a noncoding transcription-inducible strain, we analyze the relationship between antisense elongation and coding sense repression, nucleosome occupancy, and transcription-associated histone modifications using near-base pair resolution techniques. We show that antisense noncoding transcription leads to the deacetylation of a subpopulation of −1/+1 nucleosomes associated with increased H3K36me3. Reduced acetylation results in the decreased binding of the RSC chromatin remodeler at −1/+1 nucleosomes and subsequent sliding into the nucleosome-depleted region hindering pre-initiation complex association. Finally, we extend our model by showing that natural antisense noncoding transcription significantly represses ∼20% of S. cerevisiae genes through this chromatin-based transcription interference mechanism.</p

A novel role for Sem1 and TREX-2 in transcription involves their impact on recruitment and H2B deubiquitylation activity of SAGA

14 páginas, 7 figuras. Material suplementario disponible en: http://dx.doi.org/10.1093/nar/gkt272Transcription and mRNA export are linked processes. However, the molecular mechanisms of this coordination are not clear. Sus1 (hENY2) participates in this coordination as part of two protein complexes: SAGA, a transcriptional co-activator; TREX-2, which functions in mRNA biogenesis and export. Here, we investigate the coordinated action of SAGA and TREX-2 required for gene expression. We demonstrate that TREX-2 subunit Sem1 also participates in transcription activation. Like Sus1, Sem1 is required for the induction of ARG1 and GAL1, these being SAGA-regulated genes. Chromatin immunoprecipitations show that proper recruitment of certain SAGA subunits to the GAL1 promoter depends on Sem1. Notably, both in vivo and in vitro analyses reveal that Sem1 influences SAGA-dependent histone H2B deubiquitylation. Most of these phenotypes are also found to depend on another TREX-2 subunit, Thp1. These results unveil a new role for Sem1 in the activation of the SAGA-dependent gene GAL1 and influencing H2B deubiquitylation. Our work provides insights into a novel functional relationship between Sem1 and the SAGA complex.MINECO [BFU2011-23418; to S.R.N.]; GV [PROMETEO/2012/061 to S.R.N.]; CIPF and FPU [AP2009-0917] to E.G.O. and V.G.M., respectively programs. Funding for open access charge: MINECO [BFU2011-23418 to S.R.N.].Peer reviewe

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

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The SAGA/TREX-2 subunit Sus1 binds widely to transcribed genes and affects mRNA turnover globally

Abstract Background Eukaryotic transcription is regulated through two complexes, the general transcription factor IID (TFIID) and the coactivator Spt–Ada–Gcn5 acetyltransferase (SAGA). Recent findings confirm that both TFIID and SAGA contribute to the synthesis of nearly all transcripts and are recruited genome-wide in yeast. However, how this broad recruitment confers selectivity under specific conditions remains an open question. Results Here we find that the SAGA/TREX-2 subunit Sus1 associates with upstream regulatory regions of many yeast genes and that heat shock drastically changes Sus1 binding. While Sus1 binding to TFIID-dominated genes is not affected by temperature, its recruitment to SAGA-dominated genes and RP genes is significantly disturbed under heat shock, with Sus1 relocated to environmental stress-responsive genes in these conditions. Moreover, in contrast to recent results showing that SAGA deubiquitinating enzyme Ubp8 is dispensable for RNA synthesis, genomic run-on experiments demonstrate that Sus1 contributes to synthesis and stability of a wide range of transcripts. Conclusions Our study provides support for a model in which SAGA/TREX-2 factor Sus1 acts as a global transcriptional regulator in yeast but has differential activity at yeast genes as a function of their transcription rate or during stress conditions.VG-M was supported by the FPU program from MEC (AP2009-0917). This study was supported by funds from MINECO (BFU2011-23418 and BFU2014-57636 to SR-N, BIO2012-40244 to AC and BFU2016-77728-C3-3-P to JEP-O) and GV (PROMETEO/2012/061, PROMETEO/2016/093 and ACOMP/2014/061 to SR-N, and PROMETEOII/2015/006, ACOMP/2014/253 to JEP-O). The contributions of PF-T and AC were supported by the FP7 Project STATegra 306000. BFP was supported by NIH GM059055. BFP has a fnancial interest in Peconic, LLC, which utilizes the ChIP-exo technology implemented in this study and could potentially beneft from the outcomes of this research

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