The promiscuity of the SAGA complex subunits: Multifunctional or moonlighting proteins?

Gene expression, the decoding of DNA information into accessible instructions for protein synthesis, is a complex process in which multiple steps, including transcription, mRNA processing and mRNA export, are regulated by different factors. One of the first steps in this process involves chemical and structural changes in chromatin to allow transcription. For such changes to occur, histone tail and DNA epigenetic modifications foster the binding of transcription factors to promoter regions. The SAGA coactivator complex plays a crucial role in this process by mediating histone acetylation through Gcn5, and histone deubiquitination through Ubp8 enzymes. However, most SAGA subunits interact physically with other proteins beyond the SAGA complex. These interactions could represent SAGA-independent functions or a mechanism to widen SAGA multifunctionality. Among the different mechanisms to perform more than one function, protein moonlighting defines unrelated molecular activities for the same polypeptide sequence. Unlike pleiotropy, where a single gene can affect different phenotypes, moonlighting necessarily involves separate functions of a protein at the molecular level. In this review we describe in detail some of the alternative physical interactions of several SAGA subunits. In some cases, the alternative role constitutes a clear moonlighting function, whereas in most of them the lack of molecular evidence means that we can only define these interactions as promiscuous that require further work to verify if these are moonlighting functions.

SAGA-CORE subunit Spt7 is required for correct Ubp8 localization, chromatin association and deubiquitinase activity.

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.

Sharing Marks: H3K4 Methylation and H2B Ubiquitination as Features of Meiotic Recombination and Transcription.

Meiosis is a specialized cell division that gives raise to four haploid gametes from a single diploid cell. During meiosis, homologous recombination is crucial to ensure genetic diversity and guarantee accurate chromosome segregation. Both the formation of programmed meiotic DNA double-strand breaks (DSBs) and their repair using homologous chromosomes are essential and highly regulated pathways. Similar to other processes that take place in the context of chromatin, histone posttranslational modifications (PTMs) constitute one of the major mechanisms to regulate meiotic recombination. In this review, we focus on specific PTMs occurring in histone tails as driving forces of different molecular events, including meiotic recombination and transcription. In particular, we concentrate on the influence of H3K4me3, H2BK123ub, and their corresponding molecular machineries that write, read, and erase these histone marks. The Spp1 subunit within the Complex of Proteins Associated with Set1 (COMPASS) is a critical regulator of H3K4me3-dependent meiotic DSB formation. On the other hand, the PAF1c (RNA polymerase II associated factor 1 complex) drives the ubiquitination of H2BK123 by Rad6-Bre1. We also discuss emerging evidence obtained by cryo-electron microscopy (EM) structure determination that has provided new insights into how the "cross-talk" between these two marks is accomplished.

A multi-omics dataset of heat-shock response in the yeast RNA binding protein Mip6.

Gene expression is a biological process regulated at different molecular levels, including chromatin accessibility, transcription, and RNA maturation and transport. In addition, these regulatory mechanisms have strong links with cellular metabolism. Here we present a multi-omics dataset that captures different aspects of this multi-layered process in yeast. We obtained RNA-seq, metabolomics, and H4K12ac ChIP-seq data for wild-type and mip6Δ strains during a heat-shock time course. Mip6 is an RNA-binding protein that contributes to RNA export during environmental stress and is informative of the contribution of post-transcriptional regulation to control cellular adaptations to environmental changes. The experiment was performed in quadruplicate, and the different omics measurements were obtained from the same biological samples, which facilitates the integration and analysis of data using covariance-based methods. We validate our dataset by showing that ChIP-seq, RNA-seq and metabolomics signals recapitulate existing knowledge about the response of ribosomal genes and the contribution of trehalose metabolism to heat stress. Raw data, processed data and preprocessing scripts are made available.

Mip6 binds directly to the Mex67 UBA domain to maintain low levels of Msn2/4 stress-dependent mRNAs.

RNA-binding proteins (RBPs) participate in all steps of gene expression, underscoring their potential as regulators of RNA homeostasis. We structurally and functionally characterize Mip6, a four-RNA recognition motif (RRM)-containing RBP, as a functional and physical interactor of the export factor Mex67. Mip6-RRM4 directly interacts with the ubiquitin-associated (UBA) domain of Mex67 through a loop containing tryptophan 442. Mip6 shuttles between the nucleus and the cytoplasm in a Mex67-dependent manner and concentrates in cytoplasmic foci under stress. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation experiments show preferential binding of Mip6 to mRNAs regulated by the stress-response Msn2/4 transcription factors. Consistent with this binding, MIP6 deletion affects their export and expression levels. Additionally, Mip6 interacts physically and/or functionally with proteins with a role in mRNA metabolism and transcription such as Rrp6, Xrn1, Sgf73, and Rpb1. These results reveal a novel role for Mip6 in the homeostasis of Msn2/4-dependent transcripts through its direct interaction with the Mex67 UBA domain.

A role for Mog1 in H2Bub1 and H3K4me3 regulation affecting RNAPII transcription and mRNA export.

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.

An exon three-way junction structure modulates splicing and degradation of the SUS1 yeast pre-mRNA.

The SUS1 gene of Saccharomyces cerevisiae is unusual as it contains two introns and undergoes alternative splicing, retaining one or both introns depending on growth conditions. The exon located between the two introns can be skipped during splicing and has been detected in circular form. This exon (E2) has also been found to influence the splicing of the flanking introns, an unusual situation in budding yeast where splicing mainly relies on intron recognition. Using SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension), NMR spectroscopy, gel electrophoresis and UV thermal denaturation experiments combined with computational predictions, we show that E2 of SUS1 comprises a conserved double-helical stem topped by a three-way junction. One of the hairpins emerging from the junction exhibited significant thermal stability and was capped by a purine-rich loop structurally related to the substrate loop of the VS ribozyme. Cellular assays revealed that three mutants containing altered E2 structures had impaired SUS1 expression, and that a compensatory mutation restoring the conserved stem recovered expression to wild-type levels. Semi-quantitative RT-PCR measurements paralleled these results, and revealed that mutations in E2 altered splicing and transcript degradation processes. Thus, exon structure plays an important role in SUS1 RNA metabolism.

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

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.

SQANTI: extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification.

High-throughput sequencing of full-length transcripts using long reads has paved the way for the discovery of thousands of novel transcripts, even in well-annotated mammalian species. The advances in sequencing technology have created a need for studies and tools that can characterize these novel variants. Here, we present SQANTI, an automated pipeline for the classification of long-read transcripts that can assess the quality of data and the preprocessing pipeline using 47 unique descriptors. We apply SQANTI to a neuronal mouse transcriptome using Pacific Biosciences (PacBio) long reads and illustrate how the tool is effective in characterizing and describing the composition of the full-length transcriptome. We perform extensive evaluation of ToFU PacBio transcripts by PCR to reveal that an important number of the novel transcripts are technical artifacts of the sequencing approach and that SQANTI quality descriptors can be used to engineer a filtering strategy to remove them. Most novel transcripts in this curated transcriptome are novel combinations of existing splice sites, resulting more frequently in novel ORFs than novel UTRs, and are enriched in both general metabolic and neural-specific functions. We show that these new transcripts have a major impact in the correct quantification of transcript levels by state-of-the-art short-read-based quantification algorithms. By comparing our iso-transcriptome with public proteomics databases, we find that alternative isoforms are elusive to proteogenomics detection. SQANTI allows the user to maximize the analytical outcome of long-read technologies by providing the tools to deliver quality-evaluated and curated full-length transcriptomes.

The evolutionarily conserved factor Sus1/ENY2 plays a role in telomere length maintenance.

Sus1 is a conserved protein involved in histone H2B de-ubiquitination and mRNA export from the nucleus in eukaryotes. Previous studies implicated Sus1 partners in genome integrity including telomere homeostasis. However, the implication of Sus1 in telomere maintenance remains largely unknown. In this study, we found that yeast Sus1 interacts physically and genetically with factors involved in telomere maintenance and its absence leads to elongated telomeres. Deletion of several of Sus1's partners also leads to longer telomeres. Our results rule out a direct role for Sus1 in recruiting telomerase subunits to telomeres. However, we observe that deletion of SUS1 leads to elongated telomeres even in the presence of mutations like sem1Δ, esc2Δ and rsc2Δ, which cause telomere shortening. We find that rsc2Δ (short telomeres) have reduced levels of mono-ubiquitinated histone H2B at lysine 123 (H2BK123ub1), whereas sus1Δ mutants or double-mutants sus1Δ rsc2Δ exhibit longer telomeres and higher H2BK123ub1 levels. These results suggest that Sus1 activity as a H2B de-ubiquitination modulator plays a role in negatively regulating telomere length. Our results provide solid evidence for a role of Sus1 in negatively regulating telomere length through the modulation of H2BK123 mono-ubiquitination and its interaction with the nuclear pore complex.

An intronic RNA structure modulates expression of the mRNA biogenesis factor Sus1.

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.

Unveiling novel interactions of histone chaperone Asf1 linked to TREX-2 factors Sus1 and Thp1.

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.

Optimised protocols for the metabolic profiling of S. cerevisiae by 1H-NMR and HRMAS spectroscopy.

An optimised extraction protocol for the analysis of Saccharomyces cerevisiae aqueous and organic metabolites by nuclear magnetic resonance spectroscopy that allows the identification and quantification of up to 50 different compounds is presented. The method was compared with other metabolic profiling protocols for S. cerevisiae, where generally different analytical techniques are applied for metabolite quantification. In addition, the analysis of intact S. cerevisiae cells by HRMAS was implemented for the first time as a complementary method. The optimised protocols were applied to study the metabolic effect of glucose and galactose on S. cerevisiae growth. Furthermore, the metabolic reaction of S. cerevisiae to osmotic stress has been studied.

The Hog1 stress-activated protein kinase targets nucleoporins to control mRNA export upon stress.

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.

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

Transcription 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.

Comparison of global responses to mild deficiency and excess copper levels in Arabidopsis seedlings.

Copper is an essential micronutrient in higher plants, but it is toxic in excess. The fine adjustments required to fit copper nutritional demands for optimal growth are illustrated by the diverse, severe symptoms resulting from copper deficiency and excess. Here, a differential transcriptomic analysis was done between Arabidopsis thaliana plants suffering from mild copper deficiency and those with a slight copper excess. The effects on the genes encoding cuproproteins or copper homeostasis factors were included in a CuAt database, which was organised to collect additional information and connections to other databases. The categories overrepresented under copper deficiency and copper excess conditions are discussed. Different members of the categories overrepresented under copper deficiency conditions were both dependent and independent of the general copper deficiency transcriptional regulator SPL7. The putative regulatory elements in the promoter of the copper deficiency overrepresented genes, particularly of the iron superoxide dismutase gene FSD1, were also analysed. A 65 base pair promoter fragment, with at least three GTAC sequences, was found to be not only characteristic of them all, but was responsible for most of the FSD1 copper-dependent regulations. Moreover, a new molecular marker for the slight excess copper nutritional status is proposed. Taken together, these data further contribute to characterise copper nutritional responses in higher plants.

The prefoldin bud27 mediates the assembly of the eukaryotic RNA polymerases in an rpb5-dependent manner.

The unconventional prefoldin URI/RMP, in humans, and its orthologue in yeast, Bud27, have been proposed to participate in the biogenesis of the RNA polymerases. However, this role of Bud27 has not been confirmed and is poorly elucidated. Our data help clarify the mechanisms governing biogenesis of the three eukaryotic RNA pols. We show evidence that Bud27 is the first example of a protein that participates in the biogenesis of the three eukaryotic RNA polymerases and the first example of a protein modulating their assembly instead of their nuclear transport. In addition we demonstrate that the role of Bud27 in RNA pols biogenesis depends on Rpb5. In fact, lack of BUD27 affects growth and leads to a substantial accumulation of the three RNA polymerases in the cytoplasm, defects offset by the overexpression of RPB5. Supporting this, our data demonstrate that the lack of Bud27 affects the correct assembly of Rpb5 and Rpb6 to the three RNA polymerases, suggesting that this process occurs in the cytoplasm and is a required step prior to nuclear import. Also, our data support the view that Rpb5 and Rpb6 assemble somewhat later than the rest of the complexes. Furthermore, Bud27 Rpb5-binding but not PFD-binding domain is necessary for RNA polymerases biogenesis. In agreement, we also demonstrate genetic interactions between BUD27, RPB5, and RPB6. Bud27 shuttles between the nucleus and the cytoplasm in an Xpo1-independent manner, and also independently of microtubule polarization and possibly independently of its association with the RNA pols. Our data also suggest that the role of Bud27 in RNA pols biogenesis is independent of the chaperone prefoldin (PFD) complex and of Iwr1. Finally, the role of URI seems to be conserved in humans, suggesting conserved mechanisms in RNA pols biogenesis.

Rtp1p is a karyopherin-like protein required for RNA polymerase II biogenesis.

The assembly and nuclear transport of RNA polymerase II (RNA pol II) are processes that require the participation of many auxiliary factors. In a yeast genetic screen, we identified a previously uncharacterized gene, YMR185w (renamed RTP1), which encodes a protein required for the nuclear import of RNA pol II. Using protein affinity purification coupled to mass spectrometry, we identified interactions between Rtp1p and members of the R2TP complex. Rtp1p also interacts, to a different extent, with several RNA pol II subunits. The pattern of interactions is compatible with a role for Rtp1p as an assembly factor that participates in the formation of the Rpb2/Rpb3 subassembly complex and its binding to the Rpb1p-containing subcomplex. Besides, Rtp1p has a molecular architecture characteristic of karyopherins, composed of HEAT repeats, and is able to interact with phenylalanine-glycine-containing nucleoporins. Our results define Rtp1p as a new component of the RNA pol II biogenesis machinery that plays roles in subunit assembly and likely in transport through the nuclear pore complex.

Sus1/ENY2: a multitasking protein in eukaryotic gene expression.

The purpose of this review is to provide a complete overview on the functions of the transcription/export factor Sus1. Sus1 is a tiny conserved factor in sequence and functions through the eukaryotic kingdom. Although it was discovered recently, research done to address the role of Sus1/ENY2 has provided in deep description of different mechanisms influencing gene expression. Initially found to interact with the transcription and mRNA export machinery in yeast, it is now clear that it has a broad role in mRNA biogenesis. Sus1 is necessary for histone H2B deubiquitination, mRNA export and gene gating. Moreover, interesting observations also suggest a link with the cytoplasmatic mRNP fate. Although the role of Sus1 in human cells is largely unknown, preliminary results suggest interesting links to pathological states that range from rare diseases to diabetes. We will describe what is known about Sus1/ENY2 in yeast and other eukaryotes and discuss some exciting open questions to be solved in the future.