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13 páginas, 5 figuras, 1 tabla. Contiene información suplementaria en: Supplementary information accompanies this paper at https://doi.org/10.1186/s13072-020-00367-3, Background: Histone H2B deubiquitination is performed by numerous deubiquitinases in eukaryotic cells including Ubp8, the catalytic subunit of the tetrameric deubiquitination module (DUBm: Ubp8; Sus1; Sgf11; Sgf73) of the Spt-Ada-Gcn5 acetyltransferase (SAGA). Ubp8 is linked to the rest of SAGA through Sgf73 and is activated by the adaptors Sus1 and Sgf11. It is unknown if DUBm/Ubp8 might also work in a SAGA-independent manner. Results: Here we report that a tetrameric DUBm is assembled independently of the SAGA-CORE components SPT7, ADA1 and SPT20. In the absence of SPT7, i.e., independent of the SAGA complex, Ubp8 and Sus1 are poorly recruited to SAGA-dependent genes and to chromatin. Notably, cells lacking Spt7 or Ada1, but not Spt20, show lower levels of nuclear Ubp8 than wild-type cells, suggesting a possible role for SAGA-CORE subunits in Ubp8 localization. Last, deletion of SPT7 leads to defects in Ubp8 deubiquitinase activity in in vivo and in vitro assays. Conclusions: Collectively, our studies show that the DUBm tetrameric structure can form without a complete intact SAGA-CORE complex and that it includes full-length Sgf73. However, subunits of this SAGA-CORE influence DUBm association with chromatin, its localization and its activity., This study was supported by funds to SR-N from the Spanish MINECO, MICIIN (BFU2014-57636, BFU2015-71978, PGC2018-099872-B-I00) and the Generalitat Valenciana (PROM/2012/061, ACOMP2014/061 and PROMETEO 2016/093). This work was supported by FEDER 2014–2020 and the Ministerio de Economia y Competitividad (MINECO) of Spain. V.G-M was supported by the FPU program from the Ministerio de Educación y Ciencia (AP2009-0917); C.C-N by the Generailtat Valenciana PROMETEO/2016/093; P.O-C by the FPI program from MINECO (BES2012-058587); and M.M-E by the GVA (Val I+D: ACIF/2015/025). The M.I-V lab was co-funded by European Regional Development Funds (ERDF) and the Horizon 2020 Framework Programme of the European Union under the grant agreement 688945 (Euro-BioImaging Prep Phase II).

220 págs más anexos, RNA binding proteins (RBPs) participate in virtually every step of gene expression, demonstrating a potential role as a quality control system contributing to RNA homeostasis. One of the proteins with an essential role in mRNA export in yeast is the evolutionary conserved Mex67 transporter. Although Mex67 binds to RNAs, it requires adaptor proteins to interact with quality-controlled mRNAs and undergo export through the nuclear pore complex. During heat shock, Hsf1 and Msn2/Msn4 transcription factors produce stress-responsive mRNAs. The export of Hsf1-dependent mRNAs depends on direct interaction with Mex67, without quality control mechanisms. However, we know less regarding the export of Msn2/Msn4-dependent transcripts, although various relatively unstudied RBPs may participate in this process. Previous yeast two-hybrid assays revealed an interaction between Mex67 and the putative RBP Mip6. Mip6 contains four RNA recognition motifs (RRMs) and has been described as a modulator of mRNA metabolism during sporulation. It was also shown as a protein whose overexpression promotes its accumulation in cytoplasmic dots producing a global repression of general translation and a toxic effect in cell growth. In this thesis, we report the functional characterization of Mip6 under optimal and stressful growth conditions through a study of the functional interaction with the RNA metabolism machinery, especially with the essential exporter Mex67, its mRNA targets and its influence in their metabolism, and the required elements for its subcellular localization including putative Mip6 domains and its possible posttranslational modifications. Mip6 physically and genetically interacts with the RNA export factor Mex67. Mip6 directly contacts with the ubiquitin-associated domain of Mex67 through tryptophan-442 of Mip6 RRM4, whose mutation leads to the slow growth of yeast cells in vivo. Mip6 shuttles between the nucleus and the cytoplasm in a Mex67-dependent manner and concentrates in cytoplasmic foci in response to insult by various stressors. Mip6 partially colocalizes with p-bodies and copurifies with Pab1 in stress granules (SGs). Cells lacking MIP6 display altered Pab1-SGs behavior. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) experiments demonstrated enriched Mip6 binding to Msn2/Msn4 dependent transcripts under stress-free conditions; however, Mip6 enrichment changes during heat shock showing a preferential binding to ribosomal protein genes (RPGs). Consistent with PAR-CLIP results, MIP6 deletion and the mutation of tryptophan-442 augment the expression levels of Msn2/Msn4 targets (HSP12 and CTT1). Deletion of both MIP6 and the RRP6 nuclear exosome component further increases HSP12 and CTT1 levels. Confocal microscopy studies established that deletion of the Mip6 RRMs leads to Mip6 nuclear retention at different extents. Moreover, the export of Mip6 is also regulated by a Crm1-independent Nuclear Export Signal (NES) in RRM4 that would be controlled by the karyopherin Msn5. These results reveal a novel role for Mip6 in mRNA homeostasis through direct interaction with Mex67 at post-transcriptional level. We envision a model in which the initial interaction of Mip6 with RNA contributes to the Mex67- and/or NES-dependent export of Mip6. This transport helps to regulate the levels of Msn2/Msn4-dependent mRNAs under stress-free conditions and also under heat stress conditions. However, in response to stressors, Mip6 accumulates in SGs aid the repression of RPG mRNAs.

17 páginas, 12 figuras., Monoubiquitination of histone H2B (to H2Bub1) is required for downstream events including histone H3 methylation, transcription, and mRNA export. The mechanisms and players regulating these events have not yet been completely delineated. Here, we show that the conserved Ran-binding protein Mog1 is required to sustain normal levels of H2Bub1 and H3K4me3 in Saccharomyces cerevisiae Mog1 is needed for gene body recruitment of Rad6, Bre1, and Rtf1 that are involved in H2B ubiquitination and genetically interacts with these factors. We provide evidence that the absence of MOG1 impacts on cellular processes such as transcription, DNA replication, and mRNA export, which are linked to H2Bub1. Importantly, the mRNA export defect in mog1Δ strains is exacerbated by the absence of factors that decrease H2Bub1 levels. Consistent with a role in sustaining H2Bub and H3K4me3 levels, Mog1 co-precipitates with components that participate in these modifications such as Bre1, Rtf1, and the COMPASS-associated factors Shg1 and Sdc1. These results reveal a novel role for Mog1 in H2B ubiquitination, transcription, and mRNA biogenesis., PO-C, JS-Q and CN-C were supported by the FPI, FPU and PROMETEO (BES-2012058587, FPU15/03862, PROMETEO 2016/093). This study was supported by funds to SR-N from the Spanish MINECO (BFU2011-23418, BFU2014-57636) and the Generalitat Valenciana (PROMETEO 2012/061 and ACOMP2014/061), and to JEP-O (BFU2016-77728-C3-3-P, PROMETEOII 2015/006).

15 páginas, 11 figuras. El fichero contiene una rectificación en la página 17, The control of mRNA biogenesis is exerted at several steps. In response to extracellular stimuli, stress-activated protein kinases (SAPK) modulate gene expression to maximize cell survival. In yeast, the Hog1 SAPK plays a key role in reprogramming the gene expression pattern required for cell survival upon osmostress by acting during transcriptional initiation and elongation. Here, we genetically show that an intact nuclear pore complex is important for cell survival and maximal expression of stress-responsive genes. The Hog1 SAPK associates with nuclear pore complex components and directly phosphorylates the Nup1, Nup2, and Nup60 components of the inner nuclear basket. Mutation of those factors resulted in a deficient export of stress-responsive genes upon stress. Association of Nup1, Nup2, and Nup60 to stress-responsive promoters occurs upon stress depending on Hog1 activity. Accordingly, STL1 gene territory is maintained at the nuclear periphery upon osmostress in a Hog1-dependent manner. Cells containing non-phosphorylatable mutants in Nup1 or Nup2 display reduced expression of stress-responsive genes. Together, proper mRNA biogenesis of stress-responsive genes requires of the coordinate action of synthesis and export machineries by the Hog1 SAPK, This work was supported by MINECO (Spanish government) Grant BFU2012-33503, the Consolider Ingenio 2010 program (Grant CSD2007-0015), the Fundación Marcelino Botín (to F. P.), and Grant BFU2011-26722 (to E. d. N.). Recipients of an ICREA Acadèmia (Generalitat de Catalunya). Supported by the MINECO Grant BFU2011-23418. Supported by Agence Nationale de la Recherche (Nucleopol and ODynRib-Jeune chercheur program) and Jeune équipe from Fondation pour la Recherche Médicale.

12 páginas, 5 figuras. Material suplementario disponible en: http://dx.doi.org/10.1261/rna.054049.115, Sus1 is a conserved protein involved in chromatin remodeling and mRNA biogenesis. Unlike most yeast genes, the SUS1 pre-mRNA of Saccharomyces cerevisiae contains two introns and is alternatively spliced, retaining one or both introns in response to changes in environmental conditions. SUS1 splicing may allow the cell to control Sus1 expression, but the mechanisms that regulate this process remain unknown. Using in silico analyses together with NMR spectroscopy, gel electrophoresis, and UV thermal denaturation experiments, we show that the downstream intron (I2) of SUS1 forms a weakly stable, 37-nucleotide stem-loop structure containing the branch site near its apical loop and the 3' splice site after the stem terminus. A cellular assay revealed that two of four mutants containing altered I2 structures had significantly impaired SUS1 expression. Semiquantitative RT-PCR experiments indicated that all mutants accumulated unspliced SUS1 pre-mRNA and/or induced distorted levels of fully spliced mRNA relative to wild type. Concomitantly, Sus1 cellular functions in histone H2B deubiquitination and mRNA export were affected in I2 hairpin mutants that inhibited splicing. This work demonstrates that I2 structure is relevant for SUS1 expression, and that this effect is likely exerted through modulation of splicing., This work has been supported by Ministerio de Economía y Competitividad (MINECO) of Spain (BFU2011-23418 and BFU2014-57636-P to S.R.-N. and BFU-2012-30770 to J.G.), Generalitat Valenciana of Spain (PROMETEO/2013/061, ACOMP/ 2014/061, and ACOMP/2015/096 to S.R.-N., ACOMP/2014/056 to J.G., and a Santiago Grisolía fellowship to A.A.Q.), and Universidad Católica de Valencia. We are grateful to the undergraduate students Chiara Boccellato, Alejandro Aparicio, and Joan Serrano from the ERASMUS, ADEIT-UV, and SIE-UPV programs for making contributions to this work

13 páginas, 7 figuras, 2 yablas, Anti-silencing function 1 (Asf1) is a conserved key eukaryotic histone H3/H4 chaperone that participates in a variety of DNA and chromatin-related processes. These include the assembly and disassembly of histones H3 and H4 from chromatin during replication, transcription, and DNA repair. In addition, Asf1 is required for H3K56 acetylation activity dependent on histone acetyltransferase Rtt109. Thus, Asf1 impacts on many aspects of DNA metabolism. To gain insights into the functional links of Asf1 with other cellular machineries, we employed mass spectrometry coupled to tandem affinity purification (TAP) to investigate novel physical interactions of Asf1. Under different TAP-MS analysis conditions, we describe a new repertoire of Asf1 physical interactions and novel Asf1 post-translational modifications as ubiquitination, methylation and acetylation that open up new ways to regulate Asf1 functions. Asf1 co-purifies with several subunits of the TREX-2, SAGA complexes, and with nucleoporins Nup2, Nup60, and Nup57, which are all involved in transcription coupled to mRNA export in eukaryotes. Reciprocally, Thp1 and Sus1 interact with Asf1. Albeit mRNA export and GAL1 transcription are not affected in asf1Δ a strong genetic interaction exists between ASF1 and SUS1. Notably, supporting a functional link between Asf1 and TREX-2, both Sus1 and Thp1 affect the levels of Asf1-dependent histone H3K56 acetylation and histone H3 and H4 incorporation onto chromatin. Additionally, we provide evidence for a role of Asf1 in histone H2B ubiquitination. This work proposes a functional link between Asf1 and TREX-2 components in histone metabolism at the vicinity of the nuclear pore complex., This work has been supported by MINECO, Spain (BFU2011-23418) and by the GV (PROMETEO/2013/061 Valencian Regional Government) grants to S.R.-N. M.P. is funded by MICINN, Spain (BFU2008-01976), and the GV (ACOMP2011/057 Valencian Regional Government). P.O.-C. and E.G.-O. are holders of a MINECO FPI grant and CIPF PhD grant respectively.