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.

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.

The mRNA export adaptor Yra1 contributes to DNA double-strand break repair through its C-box domain.

Yra1 is an mRNA export adaptor involved in mRNA biogenesis and export in S. cerevisiae. Yra1 overexpression was recently shown to promote accumulation of DNA:RNA hybrids favoring DNA double strand breaks (DSB), cell senescence and telomere shortening, via an unknown mechanism. Yra1 was also identified at an HO-induced DSB and Yra1 depletion causes defects in DSB repair. Previous work from our laboratory showed that Yra1 ubiquitination by Tom1 is important for mRNA export. Here, we found that Yra1 is also ubiquitinated by the SUMO-targeted ubiquitin ligases Slx5-Slx8 implicated in the interaction of irreparable DSB with nuclear pores. We further show that Yra1 binds an HO-induced irreparable DSB in a process dependent on resection. Importantly, a Yra1 mutant lacking the evolutionarily conserved C-box is not recruited to an HO-induced irreparable DSB and becomes lethal under DSB induction in a HO-cut reparable system. Together, the data provide evidence that Yra1 plays a crucial role in DSB repair via homologous recombination. While Yra1 sumoylation and/or ubiquitination are dispensable, the Yra1 C-box region is essential in this process.

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.

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.

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.

mRNA export and gene expression: the SAGA-TREX-2 connection.

In the gene expression field, different steps have been traditionally viewed as discrete and unconnected events. Nowadays, genetic and functional studies support the model of a coupled network of physical and functional connections to carry out mRNA biogenesis. Gene expression is a coordinated process that comprises different linked steps like transcription, RNA processing, export to the cytoplasm, translation and degradation of mRNAs. Its regulation is essential for cellular survival and can occur at many different levels. Transcription is the central function that occurs in the nucleus, and RNAPII plays an essential role in mRNA biogenesis. During transcription, nascent mRNA is associated with the mRNA-binding proteins involved in processing and export of the mRNA particle. Cells have developed a network of multi-protein complexes whose functions regulate the different factors involved both temporally and spatially. This coupling mechanism acts as a quality control to solve some of the organization problems of gene expression in vivo, where all the factors implicated ensure that mRNAs are ready to be exported and translated. In this review, we focus on the functional coupling of gene transcription and mRNA export, and place particular emphasis on the relationship between the NPC-associated complex, TREX2, and the transcription co-activator, SAGA. We have pinpointed the experimental evidence for Sus1's roles in transcription initiation, transcription elongation and mRNA export. In addition, we have reviewed other NPC-related processes such as gene gating to the nuclear envelope, the chromatin structure and the cellular context in which these processes take place. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.

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.

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

BACKGROUND: 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.