Understanding gene expression requires grasping its multi-step processes, from chromatin remodeling to mRNA export. This manuscript provides an accessible entry point for PhD students and junior postdocs beginning research in this area, using yeast as a model organism. We present a beginner-friendly overview of gene expression, emphasizing the dynamic interplay between chromatin modifications, transcription, mRNA processing, and export. Key topics include chromatin organization, with a focus on H2B ubiquitylation and H3 methylation crosstalk; transcriptional control by RNA polymerase II, including initiation, elongation, and termination; and the export of mRNAs via Mex67-Mtr2, adaptor proteins, and the TREX and TREX-2 complexes at the nuclear pore complex. Relevant examples from yeast genetics, biochemistry, and structural biology illustrate each step. This overview aims to equip new researchers with foundational knowledge and provides references to key studies, current challenges, and open questions in the regulation of gene expression.

Cells must rapidly adapt to environmental fluctuations, including heat stress, to maintain homeostasis and ensure survival. A key adaptive mechanism is transcriptional memory, which enables cells to "remember" prior stress exposure and mount a faster or more controlled transcriptional response upon reexposure. However, the molecular mechanisms underlying transcriptional memory in the heat shock response (HSR) remain incompletely understood. Here, we investigate the role of the RNA-binding protein (RBP) Mip6 in regulating transcriptional memory during heat stress in Saccharomyces cerevisiae. Using qRT-PCR and RNA-seq, we demonstrate that prior heat shock exposure dampens the activation of heat-responsive genes upon a second stress, a phenomenon more pronounced in mip6Δ mutants. Our transcriptomic analyses reveal that transcriptional memory predominantly suppresses excessive gene expression changes, fine-tuning stress responses. Moreover, we identify a functional and physical interaction between Mip6 and the histone deacetylase Rpd3, a key regulator of transcriptional memory. Loss of both Mip6 and Rpd3 results in synthetic growth defects under heat stress and misregulation of Msn2/4-dependent transcripts, implicating Mip6 as a novel player in the coordination of chromatin and RNA-binding mechanisms during transcriptional memory. Additionally, we show that transcriptional memory modulates metabolic homeostasis and proteostasis. Collectively, our findings implicate Mip6 in the coordination of transcriptional memory in the HSR and reveal a novel link between the RBP Mip6 and the chromatin modifier Rpd3 HDAC in stress adaptation. These insights provide a foundation for further exploration of transcriptional memory mechanisms across diverse stress conditions.

Cells not only adapt to environmental changes, but they also “remember” specific signals, allowing them to respond more rapidly to future stressors. This phenomenon, known as transcriptional memory, is orchestrated by a complex interplay of epigenetics, transcription regulators and RNA metabolism factors. Although transcriptional memory has been well-studied in various contexts, its role in the heat shock (HS) response of yeast remains largely unexplored. In our study, we delve into the dynamics of HS memory in wild-type yeast and a mip6 Δ mutant, which lacks the RNA-binding protein Mip6. Notably, Mip6 has been shown to influence the expression of key stress-related genes and maintain low Msn2/4-dependent transcript levels under standard conditions. Our transcriptomic analysis offers novel insights into how yeast cells remember HS exposure. We uncover striking differences in gene expression patterns depending on whether genes are induced or repressed during HS memory. Furthermore, we find that an initial 15-minute heat shock triggers a response that becomes attenuated with just 5 additional minutes of stress. While the response kinetics between memory and non-memory conditions are similar, we report a subtle but important role for Mip6 in fine-tuning transcriptional memory and adaptation to heat stress. Biochemical and genetic evidence also suggests that Mip6 cooperates with alternative survival pathways independent of MSN2/4, and functionally interacts with the Rpd3 histone deacetylase complex, a key player in transcriptional memory and the HS response. These findings open up new avenues for understanding the molecular mechanisms behind heat stress adaptation in eukaryotes. ### Competing Interest Statement The authors have declared no competing interest.

The inclusion of omic data into constraint-based modeling (CBM) has improved metabolic network characterization of biological models. However, the integration of semi-quantitative metabolomic data into CBM remains challenging. Here, we present MAMBA (Metabolic Adjustment via Multiomic Blocks Aggregation), a CBM approach that enables the use of semi-quantitative metabolomic data together with a gene-centric omic data type (e.g. transcriptomics, ChIP-seq or chromatin accessibility data, among others), and the combination of different time points and conditions. MAMBA outperformed other methods in terms of metabolic network characterization and metabolite prediction accuracy. As a case study, we applied MAMBA to a yeast multiomic dataset with time series design where two different yeast strains where exposed to heat stress. MAMBA captured known biology of heat stress in yeast and identified novel affected metabolic pathways. MAMBA was implemented as an integer linear programming (ILP) problem to guarantee efficient computation, and coded for MATLAB (freely available at github.com/ConesaLab/MAMBA).

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.

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.

NMR metabolomics data of yeast WT and mip6delta strains. Cultures were maintained at 30 C, or at 39 C for either 20 and 120 minutes.

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.

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.

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.

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 sem1 Δ, esc2 Δ and rsc2 Δ, which cause telomere shortening. We find that rsc2 Δ (short telomeres) have reduced levels of mono-ubiquitinated histone H2B at lysine 123 (H2BK123ub 1 ), whereas sus1 Δ mutants or double-mutants sus1 Δ rsc2 Δ exhibit longer telomeres and higher H2BK123ub 1 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.

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. The conserved Mog1 protein is important for H2Bub1, H3K4me3, transcription, and mRNA export. This novel role is distinct from its known ability to bind Ran‐GTP during protein transport. The conserved Mog1 protein is important for H2Bub1, H3K4me3, transcription, and mRNA export. This novel role is distinct from its known ability to bind Ran‐GTP during protein transport.

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.

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.

Corrigendum: SQANTI: extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification Manuel Tardaguila, Lorena de la Fuente, CristinaMarti, Cécile Pereira, Francisco Jose Pardo-Palacios, Hector del Risco, Marc Ferrell, Maravillas Mellado, Marissa Macchietto, Kenneth Verheggen, Mariola Edelmann, Iakes Ezkurdia, Jesus Vazquez, Michael Tress, Ali Mortazavi, Lennart Martens, Susana Rodriguez-Navarro, Victoria Moreno-Manzano, and Ana Conesa

Based on the claimed role transcript variants in conferring functional meaning and the lack of methods to study the functional implications of alternative splicing (AS) and alternative polyadenylation (APA), we have developed a new methodology called FAIR. This methodology will let to address the functional profiling of transcript and protein isoforms at a genome-wide level by using long-reads technologies. Moreover, we have implemented it in a software called Transcript2GO. Therefore, using PacBio and Illumina data, FAIR can generate functional hypothesis about the role of alternative isoforms in our system. First, FAIR allows the functional annotation of each PacBio-resolved isoform which involves the ORF prediction and the annotation of several functional layers: miRNA binding sites, PFAM domains, post-translational modifications, UTR motifs, NMD prediction, repetitive elements, etc. Finally, it applies different statistical methods which combine both expression data and functional annotation over each PacBio-resolved isoform. Among the several included statistical methods, we can highlight the Feature Differential Splicing which is able to point out functional elements affected by AS/APA. Using our rich annotation pipeline over a neural differentiation system, we found that nearly all genes expressing several isoforms have them annotated with at least one differential functional label, suggesting that functional profiling at isoform resolution is meaningful. We identified several functions enriched in genes regulated by differential splicing, as well as specific features as miRNAs regulated by AS/APA across conditions. Other functional insights of the relationship between function and differential splicing are easily revealed by the tools implemented in Transcript2GO.

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.

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.