RESUMEN

In Pseudomonas aeruginosa, alginate biosynthesis gene expression is inhibited by the transmembrane anti-sigma factor MucA, which sequesters the AlgU sigma factor. Cell envelope stress initiates cleavage of the MucA periplasmic domain by site-1 protease AlgW, followed by further MucA degradation to release AlgU. However, after colonizing the lungs of people with cystic fibrosis, P. aeruginosa converts to a mucoid form that produces alginate constitutively. Mucoid isolates often have mucA mutations, with the most common being mucA22, which truncates the periplasmic domain. MucA22 is degraded constitutively, and genetic studies suggested that the Prc protease is responsible. Some studies also suggested that Prc contributes to induction in strains with wild-type MucA, whereas others suggested the opposite. However, missing from all previous studies is a demonstration that Prc cleaves any protein directly, which leaves open the possibility that the effect of a prc null mutation is indirect. To address the ambiguities and shortfalls, we reevaluated the roles of AlgW and Prc as MucA and MucA22 site-1 proteases. In vivo analyses using three different assays and two different inducing conditions all suggested that AlgW is the only site-1 protease for wild-type MucA in any condition. In contrast, genetics suggested that AlgW or Prc act as MucA22 site-1 proteases in inducing conditions, whereas Prc is the only MucA22 site-1 protease in non-inducing conditions. For the first time, we also show that Prc is unable to degrade the periplasmic domain of wild-type MucA but does degrade the mutated periplasmic domain of MucA22 directly. IMPORTANCE: After colonizing the lungs of individuals with cystic fibrosis, Pseudomonas aeruginosa undergoes mutagenic conversion to a mucoid form, worsening the prognosis. Most mucoid isolates have a truncated negative regulatory protein MucA, which leads to constitutive production of the extracellular polysaccharide alginate. The protease Prc has been implicated, but not shown, to degrade the most common MucA variant, MucA22, to trigger alginate production. This work provides the first demonstration that the molecular mechanism of Prc involvement is direct degradation of the MucA22 periplasmic domain and perhaps other truncated MucA variants as well. MucA truncation and degradation by Prc might be the predominant mechanism of mucoid conversion in cystic fibrosis infections, suggesting that Prc activity could be a useful therapeutic target.

RESUMEN

Since double-stranded RNA (dsRNA) is effective for silencing a wide variety of genes, all genes are typically considered equivalent targets for such RNA interference (RNAi). Yet, loss of some regulators of RNAi in the nematode Caenorhabditis elegans can selectively impair the silencing of some genes. Here, we show that such selective requirements can be explained by an intersecting network of regulators acting on genes with differences in their RNA metabolism. In this network, the Maelstrom domain-containing protein RDE-10, the intrinsically disordered protein MUT-16, and the Argonaute protein NRDE-3 work together so that any two are required for silencing one somatic gene, but each is singly required for silencing another somatic gene, where only the requirement for NRDE-3 can be overcome by enhanced dsRNA processing. Quantitative models and their exploratory simulations led us to find that (1) changing cis-regulatory elements of the target gene can reduce the dependence on NRDE-3, (2) animals can recover from silencing in non-dividing cells, and (3) cleavage and tailing of mRNAs with UG dinucleotides, which makes them templates for amplifying small RNAs, are enriched within 'pUG zones' matching the dsRNA. Similar crosstalk between pathways and restricted amplification could result in apparently selective silencing by endogenous RNAs.

---

A variety of diseases are treated with therapeutics that switch off genes via a mechanism called RNA interference (or RNAi for short). Each gene has the instructions cells need to build a particular protein. To achieve this, the DNA sequence must first be copied into a single-stranded mRNA molecule than can be translated into protein. RNAi interferes with this process by generating a double-stranded RNA molecule which contains the same DNA sequence as the gene being turned off. A set of proteins (known as regulators) then progressively trigger a series of events that allow the double-stranded RNA to be processed into short pieces that interact with the mRNA and target it for degradation. While cells use RNAi to regulate the expression of their own genes, researchers can also artificially switch off genes by synthesizing double-stranded RNA molecules in the laboratory. However, some genes are trickier to turn off than others, and why this happens is poorly understood. To investigate, Knudsen-Palmer et al. studied how two genes (bli-1 and unc-22) are switched off by RNAi in the roundworm Caenorhabditis elegans. A previous study discovered that bli-1 and unc-22 require different regulators for their expression to be disrupted. Knudsen-Palmer et al. found that this was because the RNAi process involves an intersecting network of multiple regulators, rather than a linear pathway of regulators working one after the other. For example, bli-1 requires three regulators (MUT-16, RDE-10 and NRDE-3), whereas unc-22 only needs any two of these regulators to be switched off. Further experiments revealed that which regulators are required depends on how the gene being silenced is naturally regulated in the cell. Analysis through a computational model showed that the regulators needed for RNAi could be altered in many ways, including by changing the regions that regulators bind to on the mRNA of the target gene. These findings provide new insights into why some genes respond differently to double-stranded RNA molecules. They also suggest that testing how natural regulation of a target gene influences its response to the RNAi process could potentially lead to better therapeutics.

Asunto(s)

Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Interferencia de ARN , ARN Bicatenario , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , ARN Bicatenario/metabolismo , ARN Bicatenario/genética , Proteínas de Unión al ARN

RESUMEN

Mobile clustered regularly interspaced palindromic repeats interference (Mobile-CRISPRi) is an established method for bacterial gene expression knockdown. The deactivated Cas9 protein and guide RNA are isopropyl ß-D-1-thiogalactopyranoside inducible, and all components are integrated into the chromosome via Tn7 transposition. Here, we optimized methods specific for applying Mobile-CRISPRi in multiple Vibrio species.

RESUMEN

Kaposi's sarcoma herpesvirus (KSHV) ORF34 plays a significant role as a component of the viral pre-initiation complex (vPIC), which is indispensable for late gene expression across beta- and gammaherpesviruses. Although the key role of ORF34 within the vPIC and its function as a hub protein have been recognized, further clarification regarding its specific contribution to vPIC functionality and interactions with other components is required. This study employed a deep learning algorithm-assisted structural model of ORF34, revealing highly conserved amino acid residues across human beta- and gammaherpesviruses localized in structured domains. Thus, we engineered ORF34 alanine-scanning mutants by substituting conserved residues with alanine. These mutants were evaluated for their ability to interact with other vPIC factors and restore viral production in cells harboring the ORF34-deficient KSHV-BAC. Our experimental results highlight the crucial role of the four cysteine residues conserved in ORF34: a tetrahedral arrangement consisting of a pair of C-Xn-C consensus motifs. This suggests the potential incorporation of metal cations in interacting with ORF24 and ORF66 vPIC components, facilitating late gene transcription, and promoting overall virus production by capturing metal cations. In summary, our findings underline the essential role of conserved cysteines in KSHV ORF34 for effective vPIC assembly and viral replication, thereby enhancing our understanding of the complex interplay between the vPIC components. IMPORTANCE: The initiation of late gene transcription is universally conserved across the beta- and gammaherpesvirus families. This process employs a viral pre-initiation complex (vPIC), which is analogous to a cellular PIC. Although KSHV ORF34 is a critical factor for viral replication and is a component of the vPIC, the specifics of vPIC formation and the essential domains crucial for its function remain unclear. Structural predictions suggest that the four conserved cysteines (C170, C175, C256, and C259) form a tetrahedron that coordinates the metal cation. We investigated the role of these conserved amino acids in interactions with other vPIC components, late gene expression, and virus production to demonstrate for the first time that these cysteines are pivotal for such functions. This discovery not only deepens our comprehensive understanding of ORF34 and vPIC dynamics but also lays the groundwork for more detailed studies on herpesvirus replication mechanisms in future research.

Asunto(s)

Cisteína , Herpesvirus Humano 8 , Proteínas Virales , Replicación Viral , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Humanos , Proteínas Virales/metabolismo , Proteínas Virales/genética , Proteínas Virales/química , Cisteína/metabolismo , Cisteína/genética , Secuencia Conservada , Regulación Viral de la Expresión Génica , Células HEK293 , Secuencia de Aminoácidos

RESUMEN

Filamentous fungi can produce raw-starch-degrading enzyme, however, regulation of production of raw-starch-degrading enzyme remains poorly understood thus far. Here, two novel transcription factors raw-starch-degrading enzyme regulator D (RsrD) and raw-starch-degrading enzyme regulator E (RsrE) were identified to participate in the production of raw-starch-degrading enzyme in Penicillium oxalicum. Individual knockout of rsrD and rsrE in the parental strain Δku70 resulted in 31.1%-92.9% reduced activity of raw-starch-degrading enzyme when cultivated in the presence of commercial starch from corn. RsrD and RsrE contained a basic leucine zipper and a Zn2Cys6-type DNA-binding domain, respectively, but with unknown functions. RsrD and RsrE dynamically regulated the expression of genes encoding major amylases over time, including raw-starch-degrading glucoamylase gene PoxGA15A and α-amylase gene amy13A. Interestingly, RsrD and RsrE regulated each other at transcriptional level, through binding to their own promoter regions; nevertheless, both failed to bind to the promoter regions of PoxGA15A and amy13A, as well as the known regulatory genes for regulation of amylase gene expression. RsrD appears to play an epistatic role in the module RsrD-RsrE on regulation of amylase gene expression. This study reveals a novel regulatory pathway of fungal production of raw-starch-degrading enzyme.IMPORTANCETo survive via combating with complex extracellular environment, filamentous fungi can secrete plant polysaccharide-degrading enzymes that can efficiently hydrolyze plant polysaccharide into glucose or other mono- and disaccharides, for their nutrients. Among the plant polysaccharide-degrading enzymes, raw-starch-degrading enzymes directly degrade and convert hetero-polymeric starch into glucose and oligosaccharides below starch gelatinization temperature, which can be applied in industrial biorefinery to save cost. However, the regulatory mechanism of production of raw-starch-degrading enzyme in fungi remains unknown thus far. Here, we showed that two novel transcription factors raw-starch-degrading enzyme regulator D (RsrD) and raw-starch-degrading enzyme regulator E (RsrE) positively regulate the production of raw-starch-degrading enzyme by Penicillium oxalicum. RsrD and RsrE indirectly control the expression of genes encoding enzymes with amylase activity but directly regulate each other at transcriptional level. These findings expand diversity of gene expression regulation in fungi.

Asunto(s)

Proteínas Fúngicas , Regulación Fúngica de la Expresión Génica , Penicillium , Almidón , Factores de Transcripción , Penicillium/genética , Penicillium/enzimología , Penicillium/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Almidón/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Amilasas/metabolismo , Amilasas/genética , Regiones Promotoras Genéticas

RESUMEN

It has been proposed that inhaled EP4-receptor agonists could represent an new class of bronchodilators for the treatment of asthma that are as effective as ß2-adrenoceptor agonists. However, the genomic impact of such drugs is unknown despite being potentially deleterious to respiratory health. Herein, we used mRNA-seq to compare the transcriptomic responses produced by ONO-AE1-329 (an EP4-receptor agonist) and vilanterol (a ß2-adrenoceptor agonist) in BEAS-2B human airway epithelial cells. We also determined if an increase in cAMP mediated by different GPCRs promoted distinct transcriptional signatures by expanding this enquiry to include the adenosine A2B- and I-prostanoid receptor agonists, Bay-60-6583 and taprostene, respectively. Maximally-effective concentrations of ONO-AE1-329 and vilanterol significantly regulated (q{less than or equal to}0.05; {greater than or equal to}1.5-/{less than or equal to}0.67-fold) 232 and 320 genes, respectively of which 217 were shared. Spearman analysis showed these gene expression changes to be highly rank order correlated indicating that the functional overlap between the two interventions should be considerable. Unexpectedly, the genomic effects of ONO-AE1-329, vilanterol, Bay 60-6583 and taprostene were also highly rank order correlated. This finding raises the prospect that cAMP generated by any GPCR would initiate the same transcriptional program. Nevertheless, relative to vilanterol, ONO-AE1-329 typically behaved as a partial agonist that varied across transcripts. These data indicate that each ONO-AE1-329-regulated gene differs in sensitivity to cAMP and is defined by a unique receptor occupancy-response relationship. Moreover, if this relatively modest genomic response in BEAS-2B cells is retained in vivo, then inhaled EP4-receptor agonists could represent an alternative, and possibly safer, class of bronchodilators. Significance Statement The genomic consequences of ß2-adrenoceptor agonists in asthma are often overlooked despite being potentially harmful to lung health. We determined that ONO-AE1-329, an EP4-receptor agonist and effective bronchodilator, produced gene expression changes in BEAS-2B cells that were typically modest relative to the ß2-adrenoceptor agonist, vilanterol. Furthermore, ONO-AE1-329 behaved as a partial agonist that varied across transcripts. If this genomic activity is reproduced in vivo, then EP4-receptor agonists could represent an alternative, and possibly safer, class of bronchodilators.

RESUMEN

BACKGROUND: Organisms frequently experience environmental stresses that occur in predictable patterns and combinations. For wild Saccharomyces cerevisiae yeast growing in natural environments, cells may experience high osmotic stress when they first enter broken fruit, followed by high ethanol levels during fermentation, and then finally high levels of oxidative stress resulting from respiration of ethanol. Yeast have adapted to these patterns by evolving sophisticated "cross protection" mechanisms, where mild 'primary' doses of one stress can enhance tolerance to severe doses of a different 'secondary' stress. For example, in many yeast strains, mild osmotic or mild ethanol stresses cross protect against severe oxidative stress, which likely reflects an anticipatory response important for high fitness in nature. RESULTS: During the course of genetic mapping studies aimed at understanding the mechanisms underlying natural variation in ethanol-induced cross protection against H2O2, we found that a key H2O2 scavenging enzyme, cytosolic catalase T (Ctt1p), was absolutely essential for cross protection in a wild oak strain. This suggested the absence of other compensatory mechanisms for acquiring H2O2 resistance in that strain background under those conditions. In this study, we found surprising heterogeneity across diverse yeast strains in whether CTT1 function was fully necessary for acquired H2O2 resistance. Some strains exhibited partial dispensability of CTT1 when ethanol and/or salt were used as mild stressors, suggesting that compensatory peroxidases may play a role in acquired stress resistance in certain genetic backgrounds. We leveraged global transcriptional responses to ethanol and salt stresses in strains with different levels of CTT1 dispensability, allowing us to identify possible regulators of these alternative peroxidases and acquired stress resistance in general. CONCLUSIONS: Ultimately, this study highlights how superficially similar traits can have different underlying molecular foundations and provides a framework for understanding the diversity and regulation of stress defense mechanisms.

Asunto(s)

Peróxido de Hidrógeno , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Peróxido de Hidrógeno/metabolismo , Etanol/farmacología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estrés Oxidativo/efectos de los fármacos , Estrés Fisiológico/genética , Estrés Fisiológico/efectos de los fármacos , Presión Osmótica , Catalasa/metabolismo , Catalasa/genética , Variación Genética

RESUMEN

bb0616 of Borrelia burgdorferi, the Lyme disease pathogen, encodes a hypothetical protein of unknown function. In this study, we showed that BB0616 was not surface-exposed or associated with the membrane through localization analyses using proteinase K digestion and cell partitioning assays. The expression of bb0616 was influenced by a reduced pH but not by growth phases, elevated temperatures, or carbon sources during in vitro cultivation. A transcriptional start site for bb0616 was identified by using 5' rapid amplification of cDNA ends, which led to the identification of a functional promoter in the 5' regulatory region upstream of bb0616. By analyzing a bb0616-deficient mutant and its isogenic complemented counterparts, we found that the infectivity potential of the mutant was significantly attenuated. The inactivation of bb0616 displayed no effect on borrelial growth in the medium or resistance to oxidative stress, but the mutant was significantly more susceptible to osmotic stress. In addition, the production of global virulence regulators such as BosR and RpoS as well as virulence-associated outer surface lipoproteins OspC and DbpA was reduced in the mutant. These phenotypes were fully restored when gene mutation was complemented with a wild-type copy of bb0616. Based on these findings, we concluded that the hypothetical protein BB0616 is required for the optimal infectivity of B. burgdorferi, potentially by impacting B. burgdorferi virulence gene expression as well as survival of the spirochete under stressful conditions.

Asunto(s)

Proteínas Bacterianas , Borrelia burgdorferi , Regulación Bacteriana de la Expresión Génica , Enfermedad de Lyme , Borrelia burgdorferi/genética , Borrelia burgdorferi/patogenicidad , Borrelia burgdorferi/metabolismo , Animales , Ratones , Enfermedad de Lyme/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regiones Promotoras Genéticas , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Virulencia , Ratones Endogámicos C3H , Factor sigma/genética , Factor sigma/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Sitio de Iniciación de la Transcripción , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Prueba de Complementación Genética , Concentración de Iones de Hidrógeno

RESUMEN

Within the first 15 minutes of infection, herpes simplex virus 1 immediate early proteins repurpose cellular RNA polymerase (Pol II) for viral transcription. An important role of the viral-infected cell protein 27 (ICP27) is to facilitate viral pre-mRNA processing and export viral mRNA to the cytoplasm. Here, we use precision nuclear run-on followed by deep sequencing (PRO-seq) to characterize transcription of a viral ICP27 null mutant. At 1.5 and 3 hours post infection (hpi), we observed increased total levels of Pol II on the mutant viral genome and accumulation of Pol II downstream of poly A sites indicating increased levels of initiation and processivity. By 6 hpi, Pol II accumulation on specific mutant viral genes was higher than that on wild-type virus either at or upstream of poly A signals, depending on the gene. The PRO-seq profile of the ICP27 mutant on late genes at 6 hpi was similar but not identical to that caused by treatment with flavopiridol, a known inhibitor of RNA processivity. This pattern was different from PRO-seq profiles of other α gene mutants and upon inhibition of viral DNA replication with PAA. Together, these results indicate that ICP27 contributes to the repression of aberrant viral transcription at 1.5 and 3 hpi by inhibiting initiation and decreasing RNA processivity. However, ICP27 is needed to enhance processivity on most late genes by 6 hpi in a mechanism distinguishable from its role in viral DNA replication.IMPORTANCEWe developed and validated the use of a processivity index for precision nuclear run-on followed by deep sequencing data. The processivity index calculations confirm infected cell protein 27 (ICP27) induces downstream of transcription termination on certain host genes. The processivity indices and whole gene probe data implicate ICP27 in transient immediate early gene-mediated repression, a process that also requires ICP4, ICP22, and ICP0. The data indicate that ICP27 directly or indirectly regulates RNA polymerase (Pol II) initiation and processivity on specific genes at specific times post infection. These observations support specific and varied roles for ICP27 in regulating Pol II activity on viral genes in addition to its known roles in post transcriptional mRNA processing and export.

Asunto(s)

Genoma Viral , Herpesvirus Humano 1 , Proteínas Inmediatas-Precoces , Mutación , ARN Polimerasa II , Transcripción Viral , Animales , Humanos , Línea Celular , Chlorocebus aethiops , Regulación Viral de la Expresión Génica/efectos de los fármacos , Genes Virales/genética , Genoma Viral/genética , Herpes Simple/virología , Herpes Simple/genética , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiología , Proteínas Inmediatas-Precoces/deficiencia , Proteínas Inmediatas-Precoces/genética , Poli A/genética , Poli A/metabolismo , ARN Polimerasa II/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Células Vero , Transcripción Viral/efectos de los fármacos , Transcripción Viral/genética , Replicación Viral/genética

RESUMEN

BACKGROUND: Goose, descendants of migratory ancestors, have undergone extensive selective breeding, resulting in their remarkable ability to accumulate fat in the liver and exhibit a high tolerance for significant energy intake. As a result, goose offers an excellent model for studying obesity, metabolic disorders, and liver diseases in mammals. Although the impact of the three-dimensional arrangement of chromatin within the cell nucleus on gene expression and transcriptional regulation is widely acknowledged, the precise functions of chromatin architecture reorganization during fat deposition in goose liver tissues still need to be fully comprehended. RESULTS: In this study, geese exhibited more pronounced changes in the liver index and triglyceride (TG) content following the consumption of the high-fat diet (HFD) than mice without significant signs of inflammation. Additionally, we performed comprehensive analyses on 10 goose liver tissues (5 HFD, 5 normal), including generating high-resolution maps of chromatin architecture, conducting whole-genome gene expression profiling, and identifying H3K27ac peaks in the livers of geese and mice subjected to the HFD. Our results unveiled a multiscale restructuring of chromatin architecture, encompassing Compartment A/B, topologically associated domains, and interactions between promoters and enhancers. The dynamism of the three-dimensional genome architecture, prompted by the HFD, assumed a pivotal role in the transcriptional regulation of crucial genes. Furthermore, we identified genes that regulate chromatin conformation changes, contributing to the metabolic adaptation process of lipid deposition and hepatic fat changes in geese in response to excessive energy intake. Moreover, we conducted a cross-species analysis comparing geese and mice exposed to the HFD, revealing unique characteristics specific to the goose liver compared to a mouse. These chromatin conformation changes help elucidate the observed characteristics of fat deposition and hepatic fat regulation in geese under conditions of excessive energy intake. CONCLUSIONS: We examined the dynamic modifications in three-dimensional chromatin architecture and gene expression induced by an HFD in goose liver tissues. We conducted a cross-species analysis comparing that of mice. Our results contribute significant insights into the chromatin architecture of goose liver tissues, offering a novel perspective for investigating mammal liver diseases.

RESUMEN

The functional performance of immune cells relies on a complex transcriptional regulatory network. The three-dimensional structure of chromatin can affect chromatin status and gene expression patterns, and plays an important regulatory role in gene transcription. Currently available techniques for studying chromatin spatial structure include chromatin conformation capture techniques and their derivatives, chromatin accessibility sequencing techniques, and others. Additionally, the recently emerged deep learning technology can be utilized as a tool to enhance the analysis of data. In this review, we elucidate the definition and significance of the three-dimensional chromatin structure, summarize the technologies available for studying it, and describe the research progress on the chromatin spatial structure of dendritic cells, macrophages, T cells, B cells, and neutrophils.

RESUMEN

Extracellular proteases, such as chitinases secreted by Arthrobotrys oligospora (A. oligospora), play a crucial role in the process of nematode infection. However, post-transcriptional regulation of gene expression involving microRNAs (miRNAs) in A. oligospora remains scarcely described. Hereto, transcriptome sequencing was carried out to analyze the expression profiles of chitin-responsive miRNAs in A. oligospora. Based on the RNA-seq data, the differential expression of miRNAs (DEmiRNAs) in response to chitin was screened, identified and characterized in A. oligospora. Meanwhile, the potential target genes were predicted by the online tools miRanda and Targetscan, respectively. Furthermore, the interaction of DEmiRNA with it's target gene was validated by a dual-luciferase reporter assay system. Among 85 novel miRNAs identified, 25 miRNAs displayed significant differences in expression in A. oligospora in response to chitin. Gene Ontology (GO) analysis showed that the potential genes targeted by DEmiRNAs were enriched in the biological processes such as bio-degradation, extracellular components and cell cycle. KEGG analysis revealed that the target genes were mainly involved in Hippo, carbon and riboflavin metabolic pathway. Outstandingly, chitinase AOL_s00004g379, which is involved in the hydrolysis metabolic pathway of chitin, was confirmed to be a target gene of differential miR_70. These findings suggest that chitin-responsive miRNAs are involved in the regulation of cell proliferation, predator hyphae growth and chitinase expression through the mechanisms of post-transcriptional regulation, which provides a new perspective to the molecular mechanisms underlying miRNAs-mediated control of gene expression in A. oligospora.

Asunto(s)

Ascomicetos , Quitinasas , MicroARNs , Quitina , Quitinasas/genética , MicroARNs/genética

RESUMEN

Gene expression is a fundamental and highly regulated process involving a series of tightly coordinated steps, including transcription, post-transcriptional processing, translation, and post-translational modifications. A growing number of studies have revealed an additional layer of complexity in gene expression through the phenomenon of mRNA subcellular localization. mRNAs can be organized into membraneless subcellular structures within both the cytoplasm and the nucleus, but they can also targeted to membranes. In this review, we will summarize in particular our knowledge on localization of mRNAs to organelles, focusing on important regulators and available techniques for studying organellar localization, and significance of this localization in the broader context of gene expression regulation.

Asunto(s)

Regulación de la Expresión Génica , Procesamiento Postranscripcional del ARN , ARN Mensajero , ARN Mensajero/genética , ARN Mensajero/metabolismo , Humanos , Animales , Membrana Celular/metabolismo , Citoplasma/metabolismo , Citoplasma/genética , Núcleo Celular/metabolismo , Núcleo Celular/genética , Orgánulos/metabolismo , Orgánulos/genética

RESUMEN

Due to their high-quality characteristics, Chinese hamster ovary (CHO) cells have become the most widely used and reliable host cells for the production of recombinant therapeutic proteins in the biomedical field. Previous studies have shown that the m6A reader YTHDF3, which contains the YTH domain, can affect a variety of biological processes by regulating the translation and stability of target mRNAs. This study investigates the effect of YTHDF3 on transgenic CHO cells. The results indicate that stable overexpression of YTHDF3 significantly enhances recombinant protein expression without affecting host cell growth. Transcriptome sequencing indicated that several genes, including translation initiation factor, translation extension factor, and ribosome assembly factor, were upregulated in CHO cells overexpressing YTHDF3. In addition, cycloheximide experiments confirmed that YTHDF3 enhanced transgene expression by promoting translation in CHO cells. In conclusion, the findings in this study provide a novel approach for mammalian cell engineering to increase protein productivity by regulating m6A.

Asunto(s)

Cricetulus , Biosíntesis de Proteínas , Proteínas de Unión al ARN , Proteínas Recombinantes , Animales , Células CHO , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Biosíntesis de Proteínas/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Cricetinae

RESUMEN

Ribosomal proteins (r-proteins) are abundant, highly conserved, and multifaceted cellular proteins in all domains of life. Most r-proteins have RNA-binding properties and can form protein-protein contacts. Bacterial r-proteins govern the co-transcriptional rRNA folding during ribosome assembly and participate in the formation of the ribosome functional sites, such as the mRNA-binding site, tRNA-binding sites, the peptidyl transferase center, and the protein exit tunnel. In addition to their primary role in a cell as integral components of the protein synthesis machinery, many r-proteins can function beyond the ribosome (the phenomenon known as moonlighting), acting either as individual regulatory proteins or in complexes with various cellular components. The extraribosomal activities of r-proteins have been studied over the decades. In the past decade, our understanding of r-protein functions has advanced significantly due to intensive studies on ribosomes and gene expression mechanisms not only in model bacteria like Escherichia coli or Bacillus subtilis but also in little-explored bacterial species from various phyla. The aim of this review is to update information on the multiple functions of r-proteins in bacteria.

Asunto(s)

Proteínas Bacterianas , Proteínas Ribosómicas , Proteínas Ribosómicas/metabolismo , Proteínas Bacterianas/metabolismo , Ribosomas/metabolismo , Biosíntesis de Proteínas , Bacterias/metabolismo , Escherichia coli/metabolismo , ARN Ribosómico/metabolismo

RESUMEN

Theiler's murine encephalomyelitis virus (TMEV) infection has been used as a mouse model for two virus-induced organ-specific immune-mediated diseases. TMEV-induced demyelinating disease (TMEV-IDD) in the central nervous system (CNS) is a chronic inflammatory disease with viral persistence and an animal model of multiple sclerosis (MS) in humans. TMEV infection can also cause acute myocarditis with viral replication and immune cell infiltration in the heart, leading to cardiac fibrosis. Since platelets have been reported to modulate immune responses, we aimed to determine the role of platelets in TMEV infection. In transcriptome analyses of platelets, distinct sets of immune-related genes, including major histocompatibility complex (MHC) class I, were up- or downregulated in TMEV-infected mice at different time points. We depleted platelets from TMEV-infected mice by injecting them with platelet-specific antibodies. The platelet-depleted mice had significantly fewer viral antigen-positive cells in the CNS. Platelet depletion reduced the severities of TMEV-IDD and myocarditis, although the pathology scores did not reach statistical significance. Immunologically, the platelet-depleted mice had an increase in interferon (IFN)-γ production with a higher anti-TMEV IgG2a/IgG1 ratio. Thus, platelets may play roles in TMEV infection, such as gene expression, viral clearance, and anti-viral antibody isotype responses.

Asunto(s)

Esclerosis Múltiple , Miocarditis , Humanos , Ratones , Animales , Miocarditis/etiología , Miocarditis/metabolismo , Sistema Nervioso Central/metabolismo , Esclerosis Múltiple/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Enfermedad Crónica

RESUMEN

Multicellular communities of adherent bacteria known as biofilms are often detrimental in the context of a human host, making it important to study their formation and dispersal, especially in animal models. One such model is the symbiosis between the squid Euprymna scolopes and the bacterium Vibrio fischeri. Juvenile squid hatch aposymbiotically and selectively acquire their symbiont from natural seawater containing diverse environmental microbes. Successful pairing is facilitated by ciliary movements that direct bacteria to quiet zones on the surface of the squid's symbiotic light organ where V. fischeri forms a small aggregate or biofilm. Subsequently, the bacteria disperse from that aggregate to enter the organ, ultimately reaching and colonizing deep crypt spaces. Although transient, aggregate formation is critical for optimal colonization and is tightly controlled. In vitro studies have identified a variety of polysaccharides and proteins that comprise the extracellular matrix. Some of the most well-characterized matrix factors include the symbiosis polysaccharide (SYP), cellulose polysaccharide, and LapV adhesin. In this review, we discuss these components, their regulation, and other less understood V. fischeri biofilm contributors. We also highlight what is currently known about dispersal from these aggregates and host cues that may promote it. Finally, we briefly describe discoveries gleaned from the study of other V. fischeri isolates. By unraveling the complexities involved in V. fischeri's control over matrix components, we may begin to understand how the host environment triggers transient biofilm formation and dispersal to promote this unique symbiotic relationship.

Asunto(s)

Aliivibrio fischeri , Biopelículas , Animales , Humanos , Aliivibrio fischeri/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Adhesinas Bacterianas , Decapodiformes/microbiología , Simbiosis , Polisacáridos

RESUMEN

Since double-stranded RNA (dsRNA) is effective for silencing a wide variety of genes, all genes are typically considered equivalent targets for such RNA interference (RNAi). Yet, loss of some regulators of RNAi in the nematode C. elegans can selectively impair the silencing of some genes. Here we show that such selective requirements can be explained by an intersecting network of regulators acting on genes with differences in their RNA metabolism. In this network, the Maelstrom domain-containing protein RDE-10, the intrinsically disordered protein MUT-16, and the Argonaute protein NRDE-3 work together so that any two are required for silencing one somatic gene, but each is singly required for silencing another somatic gene, where only the requirement for NRDE-3 can be overcome by enhanced dsRNA processing. Quantitative models and their exploratory simulations led us to find that (1) changing cis-regulatory elements of the target gene can reduce the dependence on NRDE-3, (2) animals can recover from silencing in non-dividing cells and (3) cleavage and tailing of mRNAs with UG dinucleotides, which makes them templates for amplifying small RNAs, is enriched within 'pUG zones' matching the dsRNA. Similar crosstalk between pathways and restricted amplification could result in apparently selective silencing by endogenous RNAs.

RESUMEN

IMPORTANCE: Hallmarks of the developmental cycle of the obligate intracellular pathogenic bacterium Chlamydia are the primary differentiation of the infectious elementary body (EB) into the proliferative reticulate body (RB) and the secondary differentiation of RBs back into EBs. The mechanisms regulating these transitions remain unclear. In this report, we developed an effective novel strategy termed dependence on plasmid-mediated expression (DOPE) that allows for the knockdown of essential genes in Chlamydia. We demonstrate that GrgA, a Chlamydia-specific transcription factor, is essential for the secondary differentiation and optimal growth of RBs. We also show that GrgA, a chromosome-encoded regulatory protein, controls the maintenance of the chlamydial virulence plasmid. Transcriptomic analysis further indicates that GrgA functions as a critical regulator of all three sigma factors that recognize different promoter sets at developmental stages. The DOPE strategy outlined here should provide a valuable tool for future studies examining chlamydial growth, development, and pathogenicity.

Asunto(s)

Infecciones por Chlamydia , Chlamydia trachomatis , Humanos , Chlamydia trachomatis/metabolismo , Regulación Bacteriana de la Expresión Génica , Factores de Transcripción/metabolismo , Factor sigma/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo

RESUMEN

Osteoarthritis (OA), the most common form of joint disease, is characterized clinically by joint pain, stiffness, and deformity. OA is now considered a whole joint disease; however, the breakdown of the articular cartilage remains the major hallmark of the disease. Current treatments targeting OA symptoms have a limited impact on impeding or reversing the OA progression. Understanding the molecular and cellular mechanisms underlying OA development is a critical barrier to progress in OA therapy. Recent studies by the current authors' group and others have revealed that the nuclear factor of activated T cell 1 (NFAT1), a member of the NFAT family of transcription factors, regulates the expression of many anabolic and catabolic genes in articular chondrocytes of adult mice. Mice lacking NFAT1 exhibit normal skeletal development but display OA in both appendicular and spinal facet joints as adults. This review mainly focuses on the recent advances in the regulatory role of NFAT1 transcription factor in the activities of articular chondrocytes and its implication in the pathogenesis of OA.