ABSTRACT
The mammalian body possesses remarkable adaptability to cold exposure, involving intricate adjustments in cellular metabolism, ultimately leading to thermogenesis. However, cold-induced stress can impact immune response, primarily through noradrenaline-mediated pathways. In our study, we utilized a rat model subjected to short-term or long-term mild cold exposure to investigate systemic immune response during the cold acclimation. To provide human relevance, we included a group of regular cold swimmers in our study. Our research revealed complex relationship between cold exposure, neural signaling, immune response, and thermogenic regulation. One-day cold exposure triggered stress response, including cytokine production in white adipose tissue, subsequently activating brown adipose tissue, and inducing thermogenesis. We further studied systemic immune response, including the proportion of leukocytes and cytokines production. Interestingly, γδ T cells emerged as possible regulators in the broader systemic response, suggesting their possible contribution in the dynamic process of cold adaptation. We employed RNA-seq to gain further insights into the mechanisms by which γδ T cells participate in the response to cold. Additionally, we challenged rats exposed to cold with the Toll-like receptor 2 agonist, showing significant modulation of immune response. These findings significantly contribute to understanding of the physiological acclimation that occur in response to cold exposure.
ABSTRACT
Mitosis is a crucial stage in the cell cycle, controlled by a vast network of regulators responding to multiple internal and external factors. The fission yeast Schizosaccharomyces pombe demonstrates catastrophic mitotic phenotypes due to mutations or drug treatments. One of the factors provoking catastrophic mitosis is a disturbed lipid metabolism, resulting from, for example, mutations in the acetyl-CoA/biotin carboxylase (cut6), fatty acid synthase (fas2, also known as lsd1) or transcriptional regulator of lipid metabolism (cbf11) genes, as well as treatment with inhibitors of fatty acid synthesis. It has been previously shown that mitotic fidelity in lipid metabolism mutants can be partially rescued by ammonium chloride supplementation. In this study, we demonstrate that mitotic fidelity can be improved by multiple nitrogen sources. Moreover, this improvement is not limited to lipid metabolism disturbances but also applies to a number of unrelated mitotic mutants. Interestingly, the partial rescue is not achieved by restoring the lipid metabolism state, but rather indirectly. Our results highlight a novel role for nitrogen availability in mitotic fidelity.
Subject(s)
Lipid Metabolism , Mitosis , Nitrogen , Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Schizosaccharomyces/metabolism , Schizosaccharomyces/genetics , Nitrogen/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces pombe Proteins/genetics , Mutation/geneticsABSTRACT
Spliceosome assembly contributes an important but incompletely understood aspect of splicing regulation. Prp45 is a yeast splicing factor which runs as an extended fold through the spliceosome, and which may be important for bringing its components together. We performed a whole genome analysis of the genetic interaction network of the truncated allele of PRP45 (prp45(1-169)) using synthetic genetic array technology and found chromatin remodellers and modifiers as an enriched category. In agreement with related studies, H2A.Z-encoding HTZ1, and the components of SWR1, INO80, and SAGA complexes represented prominent interactors, with htz1 conferring the strongest growth defect. Because the truncation of Prp45 disproportionately affected low copy number transcripts of intron-containing genes, we prepared strains carrying intronless versions of SRB2, VPS75, or HRB1, the most affected cases with transcription-related function. Intron removal from SRB2, but not from the other genes, partly repaired some but not all the growth phenotypes identified in the genetic screen. The interaction of prp45(1-169) and htz1Δ was detectable even in cells with SRB2 intron deleted (srb2Δi). The less truncated variant, prp45(1-330), had a synthetic growth defect with htz1Δ at 16°C, which also persisted in the srb2Δi background. Moreover, htz1Δ enhanced prp45(1-330) dependent pre-mRNA hyper-accumulation of both high and low efficiency splicers, genes ECM33 and COF1, respectively. We conclude that while the expression defects of low expression intron-containing genes contribute to the genetic interactome of prp45(1-169), the genetic interactions between prp45 and htz1 alleles demonstrate the sensitivity of spliceosome assembly, delayed in prp45(1-169), to the chromatin environment.
Subject(s)
Introns , Phenotype , RNA Splicing , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Spliceosomes , Spliceosomes/metabolism , Spliceosomes/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Gene Expression Regulation, Fungal , RNA Splicing Factors/genetics , RNA Splicing Factors/metabolism , Histones/metabolism , Histones/geneticsABSTRACT
CSL proteins [named after the homologs CBF1 (RBP-Jκ in mice), Suppressor of Hairless and LAG-1] are conserved transcription factors found in animals and fungi. In the fission yeast Schizosaccharomyces pombe, they regulate various cellular processes, including cell cycle progression, lipid metabolism and cell adhesion. CSL proteins bind to DNA through their N-terminal Rel-like domain and central ß-trefoil domain. Here, we investigated the importance of DNA binding for CSL protein functions in fission yeast. We created CSL protein mutants with disrupted DNA binding and found that the vast majority of CSL protein functions depend on intact DNA binding. Specifically, DNA binding is crucial for the regulation of cell adhesion, lipid metabolism, cell cycle progression, long non-coding RNA expression and genome integrity maintenance. Interestingly, perturbed lipid metabolism leads to chromatin structure changes, potentially linking lipid metabolism to the diverse phenotypes associated with CSL protein functions. Our study highlights the critical role of DNA binding for CSL protein functions in fission yeast.
Subject(s)
Cell Cycle Proteins , Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Transcription Factors , Schizosaccharomyces/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces pombe Proteins/genetics , Protein Binding , Lipid Metabolism/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Cell Cycle/genetics , Gene Expression Regulation, Fungal , DNA, Fungal/metabolism , DNA, Fungal/geneticsABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0137820.].
ABSTRACT
Bacteria have evolved structured RNAs that can associate with RNA polymerase (RNAP). Two of them have been known so far-6S RNA and Ms1 RNA but it is unclear if any other types of RNAs binding to RNAP exist in bacteria. To identify all RNAs interacting with RNAP and the primary σ factors, we have established and performed native RIP-seq in Bacillus subtilis, Corynebacterium glutamicum, Streptomyces coelicolor, Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis. Besides known 6S RNAs in B. subtilis and Ms1 in M. smegmatis, we detected MTS2823, a homologue of Ms1, on RNAP in M. tuberculosis. In C. glutamicum, we discovered novel types of structured RNAs that associate with RNAP. Furthermore, we identified other species-specific RNAs including full-length mRNAs, revealing a previously unknown landscape of RNAs interacting with the bacterial transcription machinery.
Subject(s)
Bacterial Proteins , DNA-Directed RNA Polymerases , RNA, Bacterial , Sigma Factor , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Corynebacterium glutamicum/genetics , Corynebacterium glutamicum/metabolism , DNA-Directed RNA Polymerases/metabolism , DNA-Directed RNA Polymerases/genetics , Gene Expression Regulation, Bacterial , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/metabolism , Mycobacterium smegmatis/enzymology , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Nucleic Acid Conformation , RNA, Bacterial/metabolism , RNA, Bacterial/genetics , RNA, Untranslated , Sigma Factor/metabolism , Sigma Factor/genetics , Streptomyces coelicolor/genetics , Streptomyces coelicolor/metabolism , Transcription, GeneticABSTRACT
The fission yeast Schizosaccharomyces pombe is a popular model organism in molecular biology and cell physiology. With its ease of genetic manipulation and growth, supported by in-depth functional annotations in the PomBase database and genome-wide metabolic models,S. pombe is an attractive option for synthetic biology applications. However,S. pombe currently lacks modular tools for generating genetic circuits with more than 1 transcriptional unit. We developed a toolkit to address this gap. Adapted from the MoClo-YTK plasmid kit for Saccharomyces cerevisiae and using the same modular cloning grammar, our POMBOX toolkit is designed to facilitate fast, efficient, and modular construction of genetic circuits inS. pombe. It allows for interoperability when working with DNA sequences that are functional in bothS. cerevisiae and S. pombe (e.g., protein tags, antibiotic resistance cassettes, and coding sequences). Moreover, POMBOX enables the modular assembly of multigene pathways and increases the possible pathway length from 6 to 12 transcriptional units. We also adapted the stable integration vector homology arms to Golden Gate assembly and tested the genomic integration success rates depending on different sequence sizes, from 4 to 24 kb. We included 14 S. pombe promoters that we characterized using two fluorescent proteins, in both minimally defined (EMM2âEdinburgh minimal media) and complex (YESâyeast extract with supplements) media. Then, we examined the efficacy of 6 S. cerevisiae and 6 synthetic terminators in S. pombe. Finally, we used the POMBOX kit for a synthetic biology application in metabolic engineering and expressed plant enzymes in S. pombe to produce specialized metabolite precursors, namely, methylxanthine, amorpha-4,11-diene, and cinnamic acid from the purine, mevalonate, and aromatic amino acid pathways.
Subject(s)
Schizosaccharomyces , Schizosaccharomyces/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Synthetic Biology , Plasmids/genetics , Cloning, MolecularABSTRACT
Mitotic fidelity is crucial for the faithful distribution of genetic information into the daughter cells. Many fungal species, including the fission yeast Schizosaccharomyces pombe, undergo a closed form of mitosis, during which the nuclear envelope does not break down. In S. pombe, numerous processes have been identified that contribute to successful completion of mitosis. Notably, perturbations of lipid metabolism can lead to catastrophic mitosis and the 'cut' phenotype. It has been suggested that these mitotic defects are caused by insufficient membrane phospholipid supply during the anaphase nuclear expansion. However, it is not clear whether additional factors are involved. In this study, we characterized in detail mitosis in an S. pombe mutant lacking the Cbf11 transcription factor, which regulates lipid metabolism genes. We show that in cbf11Δ cells mitotic defects have already appeared prior to anaphase, before the nuclear expansion begins. Moreover, we identify altered cohesin dynamics and centromeric chromatin structure as additional factors affecting mitotic fidelity in cells with disrupted lipid homeostasis, providing new insights into this fundamental biological process.
Subject(s)
Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Schizosaccharomyces pombe Proteins/metabolism , Lipid Metabolism/genetics , Schizosaccharomyces/metabolism , Mitosis/genetics , CohesinsABSTRACT
Oxidative stress is associated with cardiovascular and neurodegenerative diseases, diabetes, cancer, psychiatric disorders and aging. In order to counteract, eliminate and/or adapt to the sources of stress, cells possess elaborate stress-response mechanisms, which also operate at the level of regulating transcription. Interestingly, it is becoming apparent that the metabolic state of the cell and certain metabolites can directly control the epigenetic information and gene expression. In the fission yeast Schizosaccharomyces pombe, the conserved Sty1 stress-activated protein kinase cascade is the main pathway responding to most types of stresses, and regulates the transcription of hundreds of genes via the Atf1 transcription factor. Here we report that fission yeast cells defective in fatty acid synthesis (cbf11, mga2 and ACC/cut6 mutants; FAS inhibition) show increased expression of a subset of stress-response genes. This altered gene expression depends on Sty1-Atf1, the Pap1 transcription factor, and the Gcn5 and Mst1 histone acetyltransferases, is associated with increased acetylation of histone H3 at lysine 9 in the corresponding gene promoters, and results in increased cellular resistance to oxidative stress. We propose that changes in lipid metabolism can regulate the chromatin and transcription of specific stress-response genes, which in turn might help cells to maintain redox homeostasis.
Subject(s)
Chromatin , Lipid Metabolism , Oxidative Stress , Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Acetyltransferases/genetics , Basic-Leucine Zipper Transcription Factors/genetics , Chromatin/metabolism , Gene Expression , Gene Expression Regulation, Fungal , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/genetics , Transcription Factors/geneticsABSTRACT
Epithelial-mesenchymal transition (EMT) is a cellular mechanism used by cancer cells to acquire migratory and stemness properties. In this study, we show, through in vitro, in vivo, and 3D culture experiments, that the mitochondrial protein LACTB manifests tumor suppressor properties in ovarian cancer. We show that LACTB is significantly down-regulated in epithelial ovarian cancer cells and clinical tissues. Re-expression of LACTB negatively effects the growth of cancer cells but not of non-tumorigenic cells. Mechanistically, we show that LACTB leads to differentiation of ovarian cancer cells and loss of their stemness properties, which is achieved through the inhibition of the EMT program and the LACTB-dependent down-regulation of Snail2/Slug transcription factor. This study uncovers a novel role of LACTB in ovarian cancer and proposes new ways of counteracting the oncogenic EMT program in this model system.
Subject(s)
Epithelial-Mesenchymal Transition , Ovarian Neoplasms , Snail Family Transcription Factors , beta-Lactamases , Female , Humans , beta-Lactamases/genetics , beta-Lactamases/metabolism , Carcinogenesis , Cell Line, Tumor , Epithelial-Mesenchymal Transition/genetics , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Snail Family Transcription Factors/genetics , Snail Family Transcription Factors/metabolismABSTRACT
During homologous recombination, Dbl2 protein is required for localisation of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments. RNA-seq analysis of dbl2Δ transcriptome showed that the dbl2 deletion results in upregulation of more than 500 loci in Schizosaccharomyces pombe. Compared with the loci with no change in expression, the misregulated loci in dbl2Δ are closer to long terminal and long tandem repeats. Furthermore, the misregulated loci overlap with antisense transcripts, retrotransposons, meiotic genes and genes located in subtelomeric regions. A comparison of the expression profiles revealed that Dbl2 represses the same type of genes as the HIRA histone chaperone complex. Although dbl2 deletion does not alleviate centromeric or telomeric silencing, it suppresses the silencing defect at the outer centromere caused by deletion of hip1 and slm9 genes encoding subunits of the HIRA complex. Moreover, our analyses revealed that cells lacking dbl2 show a slight increase of nucleosomes at transcription start sites and increased levels of methylated histone H3 (H3K9me2) at centromeres, subtelomeres, rDNA regions and long terminal repeats. Finally, we show that other proteins involved in homologous recombination, such as Fbh1, Rad51, Mus81 and Rad54, participate in the same gene repression pathway.
Subject(s)
Cell Cycle Proteins/metabolism , Gene Expression Regulation, Fungal , Gene Silencing , Homologous Recombination , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces pombe Proteins/physiology , Transcription Factors/metabolism , Cell Cycle Proteins/antagonists & inhibitors , Centromere , Histone Code , Nucleosomes/metabolism , Repressor Proteins/physiology , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/antagonists & inhibitors , Transcription Factors/antagonists & inhibitorsABSTRACT
RNase J1 is the major 5'-to-3' bacterial exoribonuclease. We demonstrate that in its absence, RNA polymerases (RNAPs) are redistributed on DNA, with increased RNAP occupancy on some genes without a parallel increase in transcriptional output. This suggests that some of these RNAPs represent stalled, non-transcribing complexes. We show that RNase J1 is able to resolve these stalled RNAP complexes by a "torpedo" mechanism, whereby RNase J1 degrades the nascent RNA and causes the transcription complex to disassemble upon collision with RNAP. A heterologous enzyme, yeast Xrn1 (5'-to-3' exonuclease), is less efficient than RNase J1 in resolving stalled Bacillus subtilis RNAP, suggesting that the effect is RNase-specific. Our results thus reveal a novel general principle, whereby an RNase can participate in genome-wide surveillance of stalled RNAP complexes, preventing potentially deleterious transcription-replication collisions.
Subject(s)
Bacillus subtilis/enzymology , Exoribonucleases/metabolism , RNA, Messenger/metabolism , Bacillus subtilis/genetics , Bacterial Proteins/metabolism , DNA-Directed RNA Polymerases/metabolism , Gene Expression Regulation, Bacterial , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Messenger/genetics , Transcription, GeneticABSTRACT
Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are used as models in lipid metabolic research or for industrial lipid production. Lipid droplets are highly dynamic storage bodies and their cellular content represents a convenient readout of the lipid metabolic state. Fluorescence microscopy is a method of choice for quantitative analysis of cellular lipid droplets, as it relies on widely available equipment and allows analysis of individual lipid droplets. Furthermore, microscopic image analysis can be automated, greatly increasing overall analysis throughput. Here, we describe an experimental and analytical workflow for automated detection and quantitative description of individual lipid droplets in three different model yeast species: the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus, and the budding yeast Saccharomyces cerevisiae. Lipid droplets are visualized with BODIPY 493/503, and cell-impermeable fluorescent dextran is added to the culture media to help identify cell boundaries. Cells are subjected to 3D epifluorescence microscopy in green and blue channels and the resulting z-stack images are processed automatically by a MATLAB pipeline. The procedure outputs rich quantitative data on cellular lipid droplet content and individual lipid droplet characteristics in a tabular format suitable for downstream analyses in major spreadsheet or statistical packages. We provide example analyses of lipid droplet content under various conditions that affect cellular lipid metabolism.
Subject(s)
Image Processing, Computer-Assisted/methods , Lipid Droplets/chemistry , Saccharomyces cerevisiae/chemistry , Saccharomycetales/chemistry , Schizosaccharomyces/chemistry , HumansABSTRACT
Ms1 is a sRNA recently found in mycobacteria and several other actinobacterial species. Ms1 interacts with the RNA polymerase (RNAP) core devoid of sigma factors, which differs from 6S RNA that binds to RNAP holoenzymes containing the primary sigma factor. Here we show that Ms1 is the most abundant non-rRNA transcript in stationary phase in Mycobacterium smegmatis. The accumulation of Ms1 stems from its high-level synthesis combined with decreased degradation. We identify the Ms1 promoter, PMs1 , and cis-acting elements important for its activity. Furthermore, we demonstrate that PNPase (an RNase) contributes to the differential accumulation of Ms1 during growth. Then, by comparing the transcriptomes of wt and ΔMs1 strains from stationary phase, we reveal that Ms1 affects the intracellular level of RNAP. The absence of Ms1 results in decreased levels of the mRNAs encoding ß and ß' subunits of RNAP, which is also reflected at the protein level. Thus, the ΔMs1 strain has a smaller pool of RNAPs available when the transcriptional demand increases. This contributes to the inability of the ΔMs1 strain to rapidly react to environmental changes during outgrowth from stationary phase.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Mycobacterium smegmatis/enzymology , Mycobacterium smegmatis/metabolism , RNA, Bacterial/metabolism , RNA, Small Untranslated/metabolism , Gene Deletion , Gene Expression Profiling , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/growth & development , RNA, Small Untranslated/geneticsABSTRACT
Every cell cycle iteration culminates with the resolution of a mitotic nucleus into a pair of daughter nuclei, which are distributed between the two daughter cells. In the fission yeast Schizosaccharomyces pombe, the faithful division of a mitotic nucleus depends on unperturbed lipogenesis. Upon genetically or chemically induced perturbation of lipid anabolism, S. pombe cells fail to separate the two daughter nuclei and subsequently initiate lethal cytokinesis resulting in the so-called "cut" terminal phenotype. Evidence supporting a critical role of lipid biogenesis in successful mitosis in S. pombe has been accumulating for almost two decades, but the exact mechanism explaining the reported observations had been elusive. Recently, several studies established a functional link between biosynthesis of structural phospholipids, nuclear membrane growth, and the fidelity of "closed" mitosis in S. pombe. These novel insights suggest a mechanistic explanation for the mitotic defects characteristic for some S. pombe mutants deficient in lipid anabolism and extend our knowledge of metabolic modulation within the context of the cell cycle. In this review, we cover the essential role of lipogenesis in "closed" mitosis, focusing mainly on S. pombe as a model system.
Subject(s)
Lipogenesis , Membrane Lipids/metabolism , Mitosis , Nuclear Envelope/metabolism , Phospholipids/metabolism , Schizosaccharomyces/growth & development , Schizosaccharomyces/metabolismABSTRACT
Fission yeast 'cut' mutants show defects in temporal coordination of nuclear division with cytokinesis, resulting in aberrant mitosis and lethality. Among other causes, the 'cut' phenotype can be triggered by genetic or chemical perturbation of lipid metabolism, supposedly resulting in shortage of membrane phospholipids and insufficient nuclear envelope expansion during anaphase. Interestingly, penetrance of the 'cut' phenotype in mutants of the transcription factor cbf11 and acetyl-coenzyme A carboxylase cut6, both related to lipid metabolism, is highly dependent on growth media, although the specific nutrient(s) affecting 'cut' occurrence is not known. In this study, we set out to identify the growth media component(s) responsible for 'cut' phenotype suppression in Δcbf11 and cut6-621 cells. We show that mitotic defects occur rapidly in Δcbf11 cells upon shift from the minimal EMM medium ('cut' suppressing) to the complex YES medium ('cut' promoting). By growing cells in YES medium supplemented with individual EMM components, we identified ammonium chloride, an efficiently utilized nitrogen source, as a specific and potent suppressor of the 'cut' phenotype in both Δcbf11 and cut6-621. Furthermore, we found that ammonium chloride boosts lipid droplet formation in wild-type cells. Our findings suggest a possible involvement of nutrient-responsive signaling in 'cut' suppression.
Subject(s)
Ammonium Chloride/pharmacology , Lipid Metabolism/drug effects , Mitosis/drug effects , Schizosaccharomyces/drug effects , Schizosaccharomyces/genetics , Acetyl-CoA Carboxylase/genetics , Ammonium Chloride/chemistry , Ammonium Chloride/metabolism , Culture Media/chemistry , Lipid Droplets/drug effects , Lipid Droplets/metabolism , Lipid Metabolism/genetics , Mitosis/genetics , Mutation , Penetrance , Phenotype , Schizosaccharomyces/growth & development , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/genetics , Transcription Factors/geneticsABSTRACT
The σI sigma factor from Bacillus subtilis is a σ factor associated with RNA polymerase (RNAP) that was previously implicated in adaptation of the cell to elevated temperature. Here, we provide a comprehensive characterization of this transcriptional regulator. By transcriptome sequencing (RNA-seq) of wild-type (wt) and σI-null strains at 37°C and 52°C, we identified â¼130 genes affected by the absence of σI Further analysis revealed that the majority of these genes were affected indirectly by σI The σI regulon, i.e., the genes directly regulated by σI, consists of 16 genes, of which eight (the dhb and yku operons) are involved in iron metabolism. The involvement of σI in iron metabolism was confirmed phenotypically. Next, we set up an in vitro transcription system and defined and experimentally validated the promoter sequence logo that, in addition to -35 and -10 regions, also contains extended -35 and -10 motifs. Thus, σI-dependent promoters are relatively information rich in comparison with most other promoters. In summary, this study supplies information about the least-explored σ factor from the industrially important model organism B. subtilisIMPORTANCE In bacteria, σ factors are essential for transcription initiation. Knowledge about their regulons (i.e., genes transcribed from promoters dependent on these σ factors) is the key for understanding how bacteria cope with the changing environment and could be instrumental for biotechnologically motivated rewiring of gene expression. Here, we characterize the σI regulon from the industrially important model Gram-positive bacterium Bacillus subtilis We reveal that σI affects expression of â¼130 genes, of which 16 are directly regulated by σI, including genes encoding proteins involved in iron homeostasis. Detailed analysis of promoter elements then identifies unique sequences important for σI-dependent transcription. This study thus provides a comprehensive view on this underexplored component of the B. subtilis transcription machinery.
Subject(s)
Bacillus subtilis/genetics , DNA-Directed RNA Polymerases/genetics , Gene Expression Regulation, Bacterial , Promoter Regions, Genetic , Sigma Factor/genetics , Transcription, Genetic , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Iron/metabolism , Operon , Regulon , TranscriptomeABSTRACT
Ribosomal protein genes (RPGs) in Saccharomyces cerevisiae are a remarkable regulatory group that may serve as a model for understanding genetic redundancy in evolutionary adaptations. Most RPGs exist as pairs of highly conserved functional paralogs with divergent untranslated regions and introns. We examined the roles of introns in strains with various combinations of intron and gene deletions in RPL22, RPL2, RPL16, RPL37, RPL17, RPS0, and RPS18 paralog pairs. We found that introns inhibited the expression of their genes in the RPL22 pair, with the RPL22B intron conferring a much stronger effect. While the WT RPL22A/RPL22B mRNA ratio was 93/7, the rpl22aΔi/RPL22B and RPL22A/rpl22bΔi ratios were >99/<1 and 60/40, respectively. The intron in RPL2A stimulated the expression of its own gene, but the removal of the other introns had little effect on expression of the corresponding gene pair. Rpl22 protein abundances corresponded to changes in mRNAs. Using splicing reporters containing endogenous intron sequences, we demonstrated that these effects were due to the inhibition of splicing by Rpl22 proteins but not by their RNA-binding mutant versions. Indeed, only WT Rpl22A/Rpl22B proteins (but not the mutants) interacted in a yeast three-hybrid system with an RPL22B intronic region between bp 165 and 236. Transcriptome analysis showed that both the total level of Rpl22 and the A/B ratio were important for maintaining the WT phenotype. The data presented here support the contention that the Rpl22B protein has a paralog-specific role. The RPL22 singleton of Kluyveromyces lactis, which did not undergo whole genome duplication, also responded to Rpl22-mediated inhibition in K. lactis cells. Vice versa, the overproduction of the K. lactis protein reduced the expression of RPL22A/B in S. cerevisiae. The extraribosomal function of of the K. lactis Rpl22 suggests that the loop regulating RPL22 paralogs of S. cerevisiae evolved from autoregulation.
Subject(s)
Genes, Fungal , Introns , Kluyveromyces/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , TranscriptomeABSTRACT
Splicing in S. cerevisiae has been shown to proceed cotranscriptionally, but the nature of the coupling remains a subject of debate. Here, we examine the effect of nineteen complex-related splicing factor Prp45 (a homolog of SNW1/SKIP) on cotranscriptional splicing. RNA-sequencing and RT-qPCR showed elevated pre-mRNA levels but only limited reduction of spliced mRNAs in cells expressing C-terminally truncated Prp45, Prp45(1-169). Assays with a series of reporters containing the AMA1 intron with regulatable splicing confirmed decreased splicing efficiency and showed the leakage of unspliced RNAs in prp45(1-169) cells. We also measured pre-mRNA accumulation of the meiotic MER2 gene, which depends on the expression of Mer1 factor for splicing. prp45(1-169) cells accumulated approximately threefold higher levels of MER2 pre-mRNA than WT cells only when splicing was induced. To monitor cotranscriptional splicing, we determined the presence of early spliceosome assembly factors and snRNP complexes along the ECM33 and ACT1 genes. We found that prp45(1-169) hampered the cotranscriptional recruitment of U2 and, to a larger extent, U5 and NTC, while the U1 profile was unaffected. The recruitment of Prp45(1-169) was impaired similarly to U5 snRNP and NTC. Our results imply that Prp45 is required for timely formation of complex A, prior to stable physical association of U5/NTC with the emerging pre-mRNA substrate. We suggest that Prp45 facilitates conformational rearrangements and/or contacts that couple U1 snRNP-recognition to downstream assembly events.