RESUMEN
In human pathogenic fungi, receiver domains from hybrid histidine kinases (hHK) have to recognize one HPt. To understand the recognition mechanism, we have assessed phosphorelay from receiver domains of five hHKs of group III, IV, V, VI, and XI to HPt from Chaetomium thermophilum and obtained the structures of Ct_HPt alone and in complex with the receiver domain of hHK group VI. Our data indicate that receiver domains phosphotransfer to Ct_HPt, show a low affinity for complex formation, and prevent a Leu-Thr switch to stabilize phosphoryl groups, also derived from the structures of the receiver domains of hHK group III and Candida albicans Sln1. Moreover, we have elucidated the envelope structure of C. albicans Ypd1 using small-angle X-ray scattering which reveals an extended flexible conformation of the long loop αD-αE which is not involved in phosphotransfer. Finally, we have analyzed the role of salt bridges in the structure of Ct_HPt alone.
Asunto(s)
Chaetomium , Proteínas Fúngicas , Histidina Quinasa , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Chaetomium/metabolismo , Chaetomium/genética , Chaetomium/enzimología , Histidina Quinasa/metabolismo , Histidina Quinasa/química , Histidina Quinasa/genética , Candida albicans/metabolismo , Candida albicans/enzimología , Fosforilación , Modelos Moleculares , Dispersión del Ángulo Pequeño , Conformación ProteicaRESUMEN
Rice straw breakdown is sluggish, which makes agricultural waste management difficult, however pretreatment procedures and cellulolytic fungi can address this issue. Through ITS sequencing, Chaetomium globosum C1, Aspergillus sp. F2, and Ascomycota sp. SM2 were identified from diverse sources. Ascomycota sp. SM2 exhibited the highest carboxymethyl cellulase (CMCase) activity (0.86 IU/mL) and filter-paper cellulase (FPase) activity (1.054 FPU/mL), while Aspergillus sp. F2 showed the highest CMCase activity (0.185 IU/mL) after various pretreatments of rice straw. These fungi thrived across a wide pH range, with Ascomycota sp. SM2 from pH 4 to 9, Aspergillus sp. F2, and Chaetomium globosum C1 thriving in alkaline conditions (pH 9). FTIR spectroscopy revealed significant structural changes in rice straw after enzymatic hydrolysis and solid-state fermentation, indicating lignin, cellulose, and hemicellulose degradation. Soil amendments with pretreated rice straw, cow manure, biochar, and these fungi increased root growth and soil nutrient availability, even under severe salt stress (up to 9.3 dS/m). The study emphasizes the need for a better understanding of Ascomycota sp. degradation capabilities and proposes that using cellulolytic fungus and pretreatment rice straw into soil amendments could mitigate salt-related difficulties and improve nutrient availability in salty soils.
Asunto(s)
Celulasa , Oryza , Suelo , Oryza/metabolismo , Suelo/química , Celulasa/metabolismo , Estrés Salino , Microbiología del Suelo , Celulosa/metabolismo , Chaetomium/metabolismo , Aspergillus/metabolismo , Hidrólisis , Concentración de Iones de Hidrógeno , Ascomicetos/metabolismo , Fermentación , Estiércol/microbiología , Carbón OrgánicoRESUMEN
Methylenetetrahydrofolate reductase (MTHFR) is a pivotal flavoprotein connecting the folate and methionine methyl cycles, catalyzing the conversion of methylenetetrahydrofolate to methyltetrahydrofolate. Human MTHFR (hMTHFR) undergoes elaborate allosteric regulation involving protein phosphorylation and S-adenosylmethionine (AdoMet)-dependent inhibition, though other factors such as subunit orientation and FAD status remain understudied due to the lack of a functional structural model. Here, we report crystal structures of Chaetomium thermophilum MTHFR (cMTHFR) in both active (R) and inhibited (T) states. We reveal FAD occlusion by Tyr361 in the T-state, which prevents substrate interaction. Remarkably, the inhibited form of cMTHFR accommodates two AdoMet molecules per subunit. In addition, we conducted a detailed investigation of the phosphorylation sites in hMTHFR, three of which were previously unidentified. Based on the structural framework provided by our cMTHFR model, we propose a possible mechanism to explain the allosteric structural transition of MTHFR, including the impact of phosphorylation on AdoMet-dependent inhibition.
Asunto(s)
Chaetomium , Metilenotetrahidrofolato Reductasa (NADPH2) , S-Adenosilmetionina , Metilenotetrahidrofolato Reductasa (NADPH2)/metabolismo , Metilenotetrahidrofolato Reductasa (NADPH2)/química , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , S-Adenosilmetionina/metabolismo , S-Adenosilmetionina/química , Regulación Alostérica , Chaetomium/enzimología , Chaetomium/metabolismo , Chaetomium/genética , Fosforilación , Humanos , Cristalografía por Rayos X , Modelos Moleculares , Flavina-Adenina Dinucleótido/metabolismo , Flavina-Adenina Dinucleótido/químicaRESUMEN
Histone acetylation modifications in filamentous fungi play a crucial role in epigenetic gene regulation and are closely linked to the transcription of secondary metabolite (SM) biosynthetic gene clusters (BGCs). Histone deacetylases (HDACs) play a pivotal role in determining the extent of histone acetylation modifications and act as triggers for the expression activity of target BGCs. The genus Chaetomium is widely recognized as a rich source of novel and bioactive SMs. Deletion of a class I HDAC gene of Chaetomium olivaceum SD-80A, g7489, induces a substantial pleiotropic effect on the expression of SM BGCs. The C. olivaceum SD-80A ∆g7489 strain exhibited significant changes in morphology, sporulation ability, and secondary metabolic profile, resulting in the emergence of new compound peaks. Notably, three polyketides (A1-A3) and one asterriquinone (A4) were isolated from this mutant strain. Furthermore, our study explored the BGCs of A1-A4, confirming the function of two polyketide synthases (PKSs). Collectively, our findings highlight the promising potential of molecular epigenetic approaches for the elucidation of novel active compounds and their biosynthetic elements in Chaetomium species. This finding holds great significance for the exploration and utilization of Chaetomium resources. KEY POINTS: ⢠Deletion of a class I histone deacetylase activated secondary metabolite gene clusters. ⢠Three polyketides and one asterriquinone were isolated from HDAC deleted strain. ⢠Two different PKSs were reported in C. olivaceum SD-80A.
Asunto(s)
Chaetomium , Histona Desacetilasas , Familia de Multigenes , Policétidos , Metabolismo Secundario , Chaetomium/genética , Chaetomium/enzimología , Chaetomium/metabolismo , Metabolismo Secundario/genética , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Policétidos/metabolismo , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Sintasas Poliquetidas/genética , Sintasas Poliquetidas/metabolismo , Vías Biosintéticas/genética , Epigénesis GenéticaRESUMEN
Using CRISPR-Cas9 technology and a microhomology-mediated end-joining repair system, we substituted genes of the gliotoxin pathway in Aspergillus fumigatus with genes responsible for chetomin biosynthesis from Chaetomium cochliodes, leading to the production of three new epipolythiodioxopiperazines (ETPs). This work represents the first successful endeavor to produce ETPs in a non-native host. Additionally, the simultaneous disruption of five genes in a single transformation marks the most extensive gene knockout event in filamentous fungi to date.
Asunto(s)
Aspergillus fumigatus , Gliotoxina , Piperazinas , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/genética , Piperazinas/química , Piperazinas/metabolismo , Gliotoxina/biosíntesis , Gliotoxina/química , Estructura Molecular , Chaetomium/metabolismo , Chaetomium/química , Sistemas CRISPR-CasRESUMEN
The disintegration of cytoplasm-to-vacuole targeting (Cvt) bodies and autophagic bodies in vacuoles is essential to the Cvt pathway and macroautophagy in yeast. Atg15 is a vacuolar lipase required for the degradation of both Cvt and autophagic bodies. However, the molecular mechanism of their degradation by Atg15 remains poorly understood. In a recent study, we showed that recombinant Chaetomium thermophilum Atg15 (CtAtg15) possesses phospholipase activity, and that this activity is significantly elevated by proteolytic cleavage at a site away from the active center. The proteolytic cleavage of CtAtg15 causes a conformational change around the active center, resulting in the active open state. Interestingly, activated CtAtg15 can degrade not only Cvt and autophagic bodies but also organelle membranes. On the basis of these results, we propose an activation mechanism by which Atg15, as an "organellase," functions only in vacuoles.
Asunto(s)
Autofagia , Vacuolas , Vacuolas/metabolismo , Autofagia/fisiología , Fosfolipasas/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Chaetomium/metabolismo , Membranas Intracelulares/metabolismoRESUMEN
The thermophilic fungus Chaetomium thermophilum has been used extensively for biochemical and high-resolution structural studies of protein complexes. However, subsequent functional analyses of these assemblies have been hindered owing to the lack of genetic tools compatible with this thermophile, which are typically suited to other mesophilic eukaryotic model organisms, in particular the yeast Saccharomyces cerevisiae. Hence, we aimed to find genes from C. thermophilum that are expressed under the control of different sugars and examine their associated 5' untranslated regions as promoters responsible for sugar-regulated gene expression. To identify sugar-regulated promoters in C. thermophilum, we performed comparative xylose- versus glucose-dependent gene expression studies, which uncovered a number of enzymes with induced expression in the presence of xylose but repressed expression in glucose-supplemented media. Subsequently, we cloned the promoters of the two most stringently regulated genes, the xylosidase-like gene (XYL) and xylitol dehydrogenase (XDH), obtained from this genome-wide analysis in front of a thermostable yellow fluorescent protein (YFP) reporter. With this, we demonstrated xylose-dependent YFP expression by both Western blotting and live-cell imaging fluorescence microscopy. Prompted by these results, we expressed the C. thermophilum orthologue of a well-characterized dominant-negative ribosome assembly factor mutant, under the control of the XDH promoter, which allowed us to induce a nuclear export defect on the pre-60S subunit when C. thermophilum cells were grown in xylose- but not glucose-containing medium. Altogether, our study identified xylose-regulatable promoters in C. thermophilum, which might facilitate functional studies of genes of interest in this thermophilic eukaryotic model organism.
Asunto(s)
Chaetomium , Azúcares , Azúcares/metabolismo , Xilosa/metabolismo , Chaetomium/genética , Chaetomium/metabolismo , Saccharomyces cerevisiae/genética , Glucosa/metabolismoRESUMEN
The DEAH-box helicase Prp43 has essential functions in pre-mRNA splicing and ribosome biogenesis, remodeling structured RNAs. To initiate unwinding, Prp43 must first accommodate a single-stranded RNA segment into its RNA binding channel. This allows translocation of the helicase on the RNA. G-patch (gp) factors activate Prp43 in its cellular context enhancing the intrinsically low ATPase and RNA unwinding activity. It is unclear how the RNA loading process is accomplished by Prp43 and how it is regulated by its substrates, ATP and RNA, and the G-patch partners. We developed single-molecule (sm) FRET reporters on Prp43 from Chaetomium thermophilum to monitor the conformational dynamics of the RNA binding channel in Prp43 in real-time. We show that the channel can alternate between open and closed conformations. Binding of Pfa1(gp) and ATP shifts the distribution of states towards channel opening, facilitating the accommodation of RNA. After completion of the loading process, the channel remains firmly closed during successive cycles of ATP hydrolysis, ensuring stable interaction with the RNA and processive translocation. Without Pfa1(gp), it remains predominantly closed preventing efficient RNA loading. Our data reveal how the ligands of Prp43 regulate the structural dynamics of the RNA binding channel controlling the initial binding of RNA.
Asunto(s)
Chaetomium , ARN Helicasas DEAD-box , ARN , Adenosina Trifosfato/metabolismo , Chaetomium/química , Chaetomium/metabolismo , ARN Helicasas DEAD-box/metabolismo , ADN Helicasas/genética , Conformación Molecular , ARN/metabolismo , ARN Helicasas/metabolismo , Empalme del ARN , Transferencia Resonante de Energía de Fluorescencia , Simulación de Dinámica MolecularRESUMEN
Chaetoglobosin A (CheA), a well-known macrocyclic alkaloid with prominently highly antimycotic, antiparasitic, and antitumor properties, is mainly produced by Chaetomium globosum. However, a limited understanding of the transcriptional regulation of CheA biosynthesis has hampered its application and commercialization in agriculture and biomedicine. Here, a comprehensive study of the CgXpp1 gene, which encodes a basic helix-loop-helix family regulator with a putative role in the regulation of fungal growth and CheA biosynthesis, was performed by employing CgXpp1-disruption and CgXpp1-complementation strategies in the biocontrol species C. globosum. The results suggest that the CgXpp1 gene could be an indirect negative regulator in CheA production. Interestingly, knockout of CgXpp1 considerably increased the transcription levels of key genes and related regulatory factors associated with the CheA biosynthetic. Disruption of CgXpp1 led to a significant reduction in spore production and attenuation of cell development, which was consistent with metabolome analysis results. Taken together, an in-depth analysis of pleiotropic regulation influenced by transcription factors could provide insights into the unexplored metabolic mechanisms associated with primary and secondary metabolite production.
Asunto(s)
Chaetomium , Metabolismo Secundario/genética , Chaetomium/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Exonucleases are often found associated with polymerase or helicase domains in the same enzyme or can function as autonomous entities to maintain genome stability. Here, we uncovered Chaetomium thermophilum RecQ family proteins that also have exonuclease activity in addition to their main helicase function. The novel exonuclease activity is separate from the helical core domain and coexists with the latter two enzymatic activities on the same polypeptide. The CtRecQ121-366 exonuclease region performs independently as an exonuclease. We describe its catalytic mechanism and biological characteristics. We demonstrate unequivocally that CtRecQ121-366 exclusively displays exonuclease activity and that this activity has a 3'-5' polarity that can both hydrolyze ssDNA and cleave dsDNA substrates. The hydrolytic activity of majority exonuclease is driven by bimetal ions, and this appears to be the case for the CtRecQ121-366 exonuclease as well. Additionally, the maximum activity of CtRecQ121-366 was observed at pH 8.0-9.0, low salt with Mg2+. The two helices in the structure, a6 and a7, play significant roles in the execution by anticipating their shape and changing essential amino acids.
Asunto(s)
Chaetomium , Exonucleasas , Exonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Helicasa del Síndrome de Werner/metabolismo , RecQ Helicasas , Chaetomium/metabolismoRESUMEN
Endophytic fungi are striking resources rich in bioactive structures with agrochemical significance. In order to maximize the opportunity of search for bioactive compounds, chemical epigenetic manipulation was introduced to enhance the structural diversity of the fungal products, and an UPLC-ESIMS and bioassay-guided separation was used to detect novel bioactive metabolites. Consequently, four previously undescribed compounds including two cyclopentenones (globosporins A and B) and two monoterpenoid indole alkaloids (globosporines C and D), as well as three known compounds, were isolated from the endophytic fungus Chaetomium globosporum of Euphorbia humifusa by exposure to a DNA methyltransferase inhibitor 5-azacytidine. Their structures including the absolute configurations were elucidated by the analysis of NMR spectroscopic data, HRESIMS, and TD-DFT-ECD calculations. The indole alkaloids (globosporines C and D) showed antimicrobial activities against three phytopathogenic microbes (Xanthomonas oryzae pv. oryzae, X. oryzae pv. oryzicola, and Pseudomonas syringae pv. lachrymans) with MICs in the range of 14-72 µg/mL. Mostly, globosporine D was proved to be potently anti-phytopathogenic against X. oryzae pv. oryzae in vitro and in vivo, which suggested that it has the potential to be developed as a candidate for the prevention of rice bacterial leaf blight. This work provides an efficient and environmentally friendly approach for expanding fungal products with agricultural importance.
Asunto(s)
Antiinfecciosos , Chaetomium , Euphorbia , Oryza , Alcaloides de Triptamina Secologanina , Agroquímicos/metabolismo , Antiinfecciosos/farmacología , Azacitidina/metabolismo , Chaetomium/metabolismo , ADN/metabolismo , Epigénesis Genética , Euphorbia/metabolismo , Alcaloides Indólicos/química , Metiltransferasas/metabolismo , Oryza/metabolismo , Enfermedades de las Plantas/microbiología , Alcaloides de Triptamina Secologanina/metabolismoRESUMEN
Filamentous marine fungi have proven to be a plentiful source of new natural products. Chaetomium, a widely distributed fungal genus in the marine environment, has gained much interest within the scientific community. In the last 20 years, many potential secondary metabolites have been detected from marine-derived Chaetomium. In this review, we attempt to provide a comprehensive summary of the natural products produced by marine-derived Chaetomium species. A total of 122 secondary metabolites that were described from 2001 to 2021 are covered. The structural diversity of the compounds, along with details of the sources and relevant biological properties are also provided, while the relationships between structures and their bioactivities are discussed. It is our expectation that this review will be of benefit to drug development and innovation.
Asunto(s)
Productos Biológicos , Chaetomium , Productos Biológicos/química , Chaetomium/química , Chaetomium/metabolismo , Desarrollo de MedicamentosRESUMEN
Chaetomium thermophilum, a eukaryotic thermophile, is an aspiring organism holding great potential for various biochemical and biotechnological applications. Prerequisite for genetic manipulation is a reliable transformation system for target genes combined with selection markers operating at the high growth temperatures of the fungus. Here, we present a detailed protocol for Chaetomium thermophilum protoplast transformation to allow stable chromosomal integration of constructs into its genome, rendering this eukaryotic thermophile a valuable resource for affinity purification of native thermostable protein complexes, like nuclear pore subcomplexes.
Asunto(s)
Chaetomium , Chaetomium/genética , Chaetomium/metabolismo , Cromatografía de Afinidad , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismoRESUMEN
Chaetomium thermophilum is an attractive eukaryotic model organism which, due to its unusually high temperature tolerance (optimal growth at 50-52 °C), has a thermostable proteome that can be exploited for biochemical, structural and biotechnological applications. Site directed gene manipulation for the expression of labeled target genes is a desirable approach to study the structure and function of thermostable proteins and their organization in complexes, which has not been established for this thermophile yet. Here, we describe the development of a homologous recombination system to epitope-tag chromosomal genes of interest in Chaetomium thermophilum with the goal to exploit the derived thermostable fusion proteins for tandem-affinity purification. This genetic approach was facilitated by the engineering of suitable strains, in which factors of the non-homologous end-joining pathway were deleted, thereby improving the efficiency of homologous integration at specific gene loci. Following this strategy, we could demonstrate that gene tagging via homologous recombination improved the yield of purified bait proteins and co-precipitated factors, paving the way for related studies in fundamental research and industrial applications.
Asunto(s)
Chaetomium , Chaetomium/genética , Chaetomium/metabolismo , Epítopos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Recombinación HomólogaRESUMEN
Activation of the GTPase Rab7/Ypt7 by its cognate guanine nucleotide exchange factor (GEF) Mon1-Ccz1 marks organelles such as endosomes and autophagosomes for fusion with lysosomes/vacuoles and degradation of their content. Here, we present a high-resolution cryogenic electron microscopy structure of the Mon1-Ccz1 complex that reveals its architecture in atomic detail. Mon1 and Ccz1 are arranged side by side in a pseudo-twofold symmetrical heterodimer. The three Longin domains of each Mon1 and Ccz1 are triangularly arranged, providing a strong scaffold for the catalytic center of the GEF. At the opposite side of the Ypt7-binding site, a positively charged and relatively flat patch stretches the Longin domains 2/3 of Mon1 and functions as a phosphatidylinositol phosphate-binding site, explaining how the GEF is targeted to membranes. Our work provides molecular insight into the mechanisms of endosomal Rab activation and serves as a blueprint for understanding the function of members of the Tri Longin domain Rab-GEF family.
Asunto(s)
Membrana Celular/metabolismo , Chaetomium/metabolismo , Proteínas Fúngicas/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Unión a GTP rab7/metabolismo , Membrana Celular/genética , Chaetomium/genética , Proteínas Fúngicas/genética , Complejos Multiproteicos/genética , Proteínas de Unión a GTP rab7/genéticaRESUMEN
Ribosomes are complex and highly conserved ribonucleoprotein assemblies catalyzing protein biosynthesis in every organism. Here we present high-resolution cryo-EM structures of the 80S ribosome from a thermophilic fungus in two rotational states, which due to increased 80S stability provide a number of mechanistic details of eukaryotic translation. We identify a universally conserved 'nested base-triple knot' in the 26S rRNA at the polypeptide tunnel exit with a bulged-out nucleotide that likely serves as an adaptable element for nascent chain containment and handover. We visualize the structure and dynamics of the ribosome protective factor Stm1 upon ribosomal 40S head swiveling. We describe the structural impact of a unique and essential m1acp3 Ψ 18S rRNA hyper-modification embracing the anticodon wobble-position for eukaryotic tRNA and mRNA translocation. We complete the eEF2-GTPase switch cycle describing the GDP-bound post-hydrolysis state. Taken together, our data and their integration into the structural landscape of 80S ribosomes furthers our understanding of protein biogenesis.
Asunto(s)
Chaetomium/metabolismo , Microscopía por Crioelectrón/métodos , Factor 2 de Elongación Peptídica/química , Biosíntesis de Proteínas , ARN Ribosómico/química , Ribosomas/química , Ribosomas/metabolismo , Chaetomium/química , Factor 2 de Elongación Peptídica/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , ARN de Transferencia/metabolismoRESUMEN
Chemical investigation on the deep-sea-derived fungus Chaetomium globosum led to the isolation of nine compounds. By extensive analyses of the 1D and 2D NMR as well as HR-ESI-MS spectra, their structures were elucidated as xylariol A (1), 1,3-dihydro-4,5,6-trihydroxy-7-methylisobenzofuran (2), epicoccone B (3), epicoccolide B (4), chaetoglobosin G (5), chaetoglobosin Fex (6), cochliodone A (7), cochliodone B (8), and chaetoviridin A (9), assorting as four phenolics (1-4), two cytochalosans (5-6), and three azaplilones (7-9). Compounds 1-3 were firstly reported from C.â globosum. Under the concentrations of 20 µg/mL, 1, 2, and 3 exhibited potent inâ vitro anti-HIV activity with the inhibition rates of 70 %, 75 %, and 88 %, respectively.
Asunto(s)
Fármacos Anti-VIH/química , Chaetomium/química , Agua de Mar/microbiología , Fármacos Anti-VIH/aislamiento & purificación , Fármacos Anti-VIH/farmacología , Línea Celular , Chaetomium/metabolismo , Genes Reporteros/efectos de los fármacos , Humanos , Espectroscopía de Resonancia Magnética , Conformación Molecular , Fenoles/química , Fenoles/aislamiento & purificación , Fenoles/farmacología , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Two new cytochalasans with a rare 6/6/5/5/7 pentacyclic ring system, named chaetoconvosins C-D (1: -2: ), together with two known congeners (3: -4: ), were isolated from the fermentation of an endophytic fungus, Chaetomium sp. SG-01, harbored in the fibrous roots of Schisandra glaucescens Diels. Their structures including the absolute configuration were elucidated by extensive spectroscopic (HRESIMS, NMR, and ECD) and X-ray crystallographic analyses. The TRAIL-resistance-overcoming activity of 1: -4: in a TRAIL-resistant HT29 colorectal cancer cell line was evaluated, which revealed that co-treatment of 1: -4: at 50 µM with TRAIL (150 ng/mL) reduced the HT29 cell viability by 19.0%, 24.1%, 17.9%, and 15.5%, respectively, compared to treatment with 1: -4: alone.
Asunto(s)
Alcaloides , Chaetomium , Chaetomium/química , Chaetomium/metabolismo , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Citocalasinas/farmacología , Citocalasinas/química , Estructura MolecularRESUMEN
A correct genome annotation is fundamental for research in the field of molecular and structural biology. The annotation of the reference genome of Chaetomium thermophilum has been reported previously, but it is essentially limited to open reading frames (ORFs) of protein coding genes and contains only a few noncoding transcripts. In this study, we identified and annotated full-length transcripts of C. thermophilum by deep RNA sequencing. We annotated 7044 coding genes and 4567 noncoding genes. Astonishingly, 23% of the coding genes are alternatively spliced. We identified 679 novel coding genes as well as 2878 novel noncoding genes and corrected the structural organization of more than 50% of the previously annotated genes. Furthermore, we substantially extended the Gene Ontology (GO) and Enzyme Commission (EC) lists, which provide comprehensive search tools for potential industrial applications and basic research. The identified novel transcripts and improved annotation will help to understand the gene regulatory landscape in C. thermophilum. The analysis pipeline developed here can be used to build transcriptome assemblies and identify coding and noncoding RNAs of other species.
Asunto(s)
Chaetomium/genética , Transcriptoma , Chaetomium/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Redes Reguladoras de Genes , Anotación de Secuencia MolecularRESUMEN
Protein-metabolite interactions play an important role in the cell's metabolism and many methods have been developed to screen them in vitro. However, few methods can be applied at a large scale and not alter biological state. Here we describe a proteometabolomic approach, using chromatography to generate cell fractions which are then analyzed with mass spectrometry for both protein and metabolite identification. Integrating the proteomic and metabolomic analyses makes it possible to identify protein-bound metabolites. Applying the concept to the thermophilic fungus Chaetomium thermophilum, we predict 461 likely protein-metabolite interactions, most of them novel. As a proof of principle, we experimentally validate a predicted interaction between the ribosome and isopentenyl adenine.