RESUMEN
Efficient termination is required for robust gene transcription. Eukaryotic organisms use a conserved exoribonuclease-mediated mechanism to terminate the mRNA transcription by RNA polymerase II (Pol II)1-5. Here we report two cryogenic electron microscopy structures of Saccharomyces cerevisiae Pol II pre-termination transcription complexes bound to the 5'-to-3' exoribonuclease Rat1 and its partner Rai1. Our structures show that Rat1 displaces the elongation factor Spt5 to dock at the Pol II stalk domain. Rat1 shields the RNA exit channel of Pol II, guides the nascent RNA towards its active centre and stacks three nucleotides at the 5' terminus of the nascent RNA. The structures further show that Rat1 rotates towards Pol II as it shortens RNA. Our results provide the structural mechanism for the Rat1-mediated termination of mRNA transcription by Pol II in yeast and the exoribonuclease-mediated termination of mRNA transcription in other eukaryotes.
Asunto(s)
Microscopía por Crioelectrón , Exorribonucleasas , ARN Polimerasa II , ARN Mensajero , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Terminación de la Transcripción Genética , Exorribonucleasas/química , Exorribonucleasas/metabolismo , Exorribonucleasas/ultraestructura , Modelos Moleculares , Unión Proteica , ARN Polimerasa II/química , ARN Polimerasa II/metabolismo , ARN Polimerasa II/ultraestructura , ARN Mensajero/biosíntesis , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/ultraestructura , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/ultraestructura , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructura , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/metabolismo , Factores de Elongación Transcripcional/ultraestructura , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/ultraestructura , Dominios Proteicos , ARN de Hongos/biosíntesis , ARN de Hongos/química , ARN de Hongos/genética , ARN de Hongos/ultraestructuraRESUMEN
Efficient DNA sample preparation from fungi with the rigid cell walls is still critical for successful polymerase chain reaction (PCR), one of the basic platforms in molecular diagnostics of fungi, especially in medical mycology. Common methods that involve different chaotropes to yield DNA samples have found a limited application for fungi. Here we describe a novel procedure for efficient production of permeable fungal cell envelopes with DNA inside as suitable templates for PCR. This procedure is facile, relies on boiling of fungal cells in aqueous solutions of selected chaotropic agents and additives and enables to remove RNA and proteins from PCR template samples. The use of chaotropic solutions containing 7 M urea, 1% sodium dodecyl sulfate (SDS), up to100 mM ammonia and/or 25 mM sodium citrate was the best option to yield highly purified DNA-containing cell envelopes from all fungal strains under study, including clinical Candida and Cryptococcusisolates. After treatment with the selected chaotropic mixtures, the fungal cell walls had undergone loosening and were no longer a barrier to release DNA in PCR as evident from electron microscopy examinations and successful target gene amplifications. Overall, the developed simple, fast, and low-cost approach to produce PCR-suitable templates in the form of DNA encased by permeable cell walls can find application in molecular diagnostics.
Asunto(s)
Pared Celular , Reacción en Cadena de la Polimerasa , ADN de Hongos/química , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , ARN de Hongos/química , ARN de Hongos/genética , ARN de Hongos/aislamiento & purificación , Pared Celular/químicaRESUMEN
RNA interference is an ancient mechanism with many regulatory roles in eukaryotic genomes, with small RNAs acting as their functional element. While there is a wide array of classes of small-RNA-producing loci, those resulting from stem-loop structures (hairpins) have received profuse attention. Such is the case of microRNAs (miRNAs), which have distinct roles in plants and animals. Fungi also produce small RNAs, and several publications have identified miRNAs and miRNA-like (mi/milRNA) hairpin RNAs in diverse fungal species using deep sequencing technologies. Despite this relevant source of information, relatively little is known about mi/milRNA features in fungi, mostly due to a lack of established criteria for their annotation. To systematically assess mi/milRNA characteristics and annotation confidence, we searched for publications describing mi/milRNA loci and re-assessed the annotations for 41 fungal species. We extracted and normalized the annotation data for 1727 reported mi/milRNA loci and determined their abundance profiles, concluding that less than half of the reported loci passed basic standards used for hairpin RNA discovery. We found that fungal mi/milRNA are generally more similar in size to animal miRNAs and were frequently associated with protein-coding genes. The compiled genomic analyses identified 25 mi/milRNA loci conserved in multiple species. Our pipeline allowed us to build a general hierarchy of locus quality, identifying more than 150 loci with high-quality annotations. We provide a centralized annotation of identified mi/milRNA hairpin RNAs in fungi which will serve as a resource for future research and advance in understanding the characteristics and functions of mi/milRNAs in fungal organisms.
Asunto(s)
MicroARNs , ARN de Hongos , Animales , ARN de Hongos/genética , ARN de Hongos/química , Regulación Fúngica de la Expresión Génica , MicroARNs/genética , Interferencia de ARN , Hongos/genéticaRESUMEN
Cellular RNAs in all three kingdoms of life are modified with diverse chemical modifications. These chemical modifications expand the topological repertoire of RNAs, and fine-tune their functions. Ribosomal RNA in yeast contains more than 100 chemically modified residues in the functionally crucial and evolutionary conserved regions. The chemical modifications in the rRNA are of three types-methylation of the ribose sugars at the C2-positionAbstract (Nm), isomerization of uridines to pseudouridines (Ψ), and base modifications such as (methylation (mN), acetylation (acN), and aminocarboxypropylation (acpN)). The modifications profile of the yeast rRNA has been recently completed, providing an excellent platform to analyze the function of these modifications in RNA metabolism and in cellular physiology. Remarkably, majority of the rRNA modifications and the enzymatic machineries discovered in yeast are highly conserved in eukaryotes including humans. Mutations in factors involved in rRNA modification are linked to several rare severe human diseases (e.g., X-linked Dyskeratosis congenita, the Bowen-Conradi syndrome and the William-Beuren disease). In this chapter, we summarize all rRNA modifications and the corresponding enzymatic machineries of the budding yeast.
Asunto(s)
ARN Ribosómico , Saccharomyces cerevisiae , Acetilación , Humanos , Metilación , Seudouridina/química , Seudouridina/metabolismo , Procesamiento Postranscripcional del ARN , ARN de Hongos/química , ARN de Hongos/metabolismo , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Sequence features of genes and their flanking regulatory regions are determinants of RNA transcript isoform expression and have been used as context-independent plug-and-play modules in synthetic biology. However, genetic context-including the adjacent transcriptional environment-also influences transcript isoform expression levels and boundaries. We used synthetic yeast strains with stochastically repositioned genes to systematically disentangle the effects of sequence and context. Profiling 120 million full-length transcript molecules across 612 genomic perturbations, we observed sequence-independent alterations to gene expression levels and transcript isoform boundaries that were influenced by neighboring transcription. We identified features of transcriptional context that could predict these alterations and used these features to engineer a synthetic circuit where transcript length was controlled by neighboring transcription. This demonstrates how positional context can be leveraged in synthetic genome engineering.
Asunto(s)
Genoma Fúngico , ARN de Hongos/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/genética , Transcripción Genética , Transcriptoma , Regiones no Traducidas 3' , Secuencia de Bases , Reordenamiento Génico , Variación Genética , ARN de Hongos/química , ARN de Hongos/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , RNA-Seq , Análisis de Secuencia de ARNRESUMEN
Pseudouridine (Ψ) is a ubiquitous RNA modification incorporated by pseudouridine synthase (Pus) enzymes into hundreds of noncoding and protein-coding RNA substrates. Here, we determined the contributions of substrate structure and protein sequence to binding and catalysis by pseudouridine synthase 7 (Pus7), one of the principal messenger RNA (mRNA) modifying enzymes. Pus7 is distinct among the eukaryotic Pus proteins because it modifies a wider variety of substrates and shares limited homology with other Pus family members. We solved the crystal structure of Saccharomyces cerevisiae Pus7, detailing the architecture of the eukaryotic-specific insertions thought to be responsible for the expanded substrate scope of Pus7. Additionally, we identified an insertion domain in the protein that fine-tunes Pus7 activity both in vitro and in cells. These data demonstrate that Pus7 preferentially binds substrates possessing the previously identified UGUAR (R = purine) consensus sequence and that RNA secondary structure is not a strong requirement for Pus7-binding. In contrast, the rate constants and extent of Ψ incorporation are more influenced by RNA structure, with Pus7 modifying UGUAR sequences in less-structured contexts more efficiently both in vitro and in cells. Although less-structured substrates were preferred, Pus7 fully modified every transfer RNA, mRNA, and nonnatural RNA containing the consensus recognition sequence that we tested. Our findings suggest that Pus7 is a promiscuous enzyme and lead us to propose that factors beyond inherent enzyme properties (e.g., enzyme localization, RNA structure, and competition with other RNA-binding proteins) largely dictate Pus7 substrate selection.
Asunto(s)
Secuencia de Aminoácidos , Sitios de Unión , Modelos Moleculares , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Dominio Catalítico , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN de Hongos/química , ARN de Hongos/genética , ARN Mensajero/química , ARN Mensajero/genética , Estrés Fisiológico , Relación Estructura-Actividad , Especificidad por Sustrato , Temperatura , TermodinámicaRESUMEN
5-Formylcytidine (f5C) is one type of post-transcriptional RNA modification, which is known at the wobble position of tRNA in mitochondria and essential for mitochondrial protein synthesis. Here, we show a method to detect f5C modifications in RNA and a transcriptome-wide f5C mapping technique, named f5C-seq. It is developed based on the treatment of pyridine borane, which can reduce f5C to 5,6-dihydrouracil, thus inducing C-to-T transition in f5C sites during PCR to achieve single-base resolution detection. More than 1000 f5C sites were identified after mapping in Saccharomyces cerevisiae by f5C-seq. Moreover, codon composition demonstrated a preference for f5C within wobble sites in mRNA, suggesting the potential role in regulation of translation. These findings expand the scope of the understanding of cytosine modifications in mRNA.
Asunto(s)
Procesamiento Postranscripcional del ARN , ARN Mensajero/química , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismo , Histona H2a Dioxigenasa, Homólogo 1 de AlkB/química , Histona H2a Dioxigenasa, Homólogo 1 de AlkB/metabolismo , Secuencia de Bases , Citidina/análogos & derivados , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Humanos , ARN de Hongos/química , ARN de Hongos/metabolismo , ARN Mensajero/genética , Saccharomyces cerevisiae/genéticaRESUMEN
In vivo characterization of RNA-protein interactions is the key for understanding RNA regulatory mechanisms. Herein, we describe a protocol for detection of proteins interacting with polyadenylated RNAs in the yeast Saccharomyces cerevisiae. Proteins are crosslinked to nucleic acids in vivo by ultraviolet (UV) irradiation of cells, and poly(A)-containing RNAs with bound proteins are isolated from cell lysates using oligo[dT]25 beads. RBPs can be detected by immunoblot analysis or with mass spectrometry to define the mRNA-binding proteome (mRBPome) and its changes under stress. For complete details on the use and execution of this protocol, please refer to Matia-González et al. (2021, 2015).
Asunto(s)
Espectrometría de Masas/métodos , ARN de Hongos , ARN Mensajero , Proteínas de Unión al ARN , Proteínas de Saccharomyces cerevisiae , Mapeo de Interacción de Proteínas , Proteoma , Proteómica , ARN de Hongos/análisis , ARN de Hongos/química , ARN de Hongos/aislamiento & purificación , ARN de Hongos/metabolismo , ARN Mensajero/análisis , ARN Mensajero/química , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Identifying how R-loops are generated is crucial to know how transcription compromises genome integrity. We show by genome-wide analysis of conditional yeast mutants that the THO transcription complex, prevents R-loop formation in G1 and S-phase, whereas the Sen1 DNA-RNA helicase prevents them only in S-phase. Interestingly, damage accumulates asymmetrically downstream of the replication fork in sen1 cells but symmetrically in the hpr1 THO mutant. Our results indicate that: R-loops form co-transcriptionally independently of DNA replication; that THO is a general and cell-cycle independent safeguard against R-loops, and that Sen1, in contrast to previously believed, is an S-phase-specific R-loop resolvase. These conclusions have important implications for the mechanism of R-loop formation and the role of other factors reported to affect on R-loop homeostasis.
Asunto(s)
ADN de Hongos/química , Estructuras R-Loop , ARN de Hongos/química , Ciclo Celular/genética , Ciclo Celular/fisiología , Daño del ADN , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN de Hongos/genética , ADN de Hongos/metabolismo , Genes Fúngicos , Inestabilidad Genómica , Modelos Biológicos , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estructuras R-Loop/genética , Estructuras R-Loop/fisiología , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN de Hongos/genética , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Many non-coding RNAs with known functions are structurally conserved: their intramolecular secondary and tertiary interactions are maintained across evolutionary time. Consequently, the presence of conserved structure in multiple sequence alignments can be used to identify candidate functional non-coding RNAs. Here, we present a bioinformatics method that couples iterative homology search with covariation analysis to assess whether a genomic region has evidence of conserved RNA structure. We used this method to examine all unannotated regions of five well-studied fungal genomes (Saccharomyces cerevisiae, Candida albicans, Neurospora crassa, Aspergillus fumigatus, and Schizosaccharomyces pombe). We identified 17 novel structurally conserved non-coding RNA candidates, which include four H/ACA box small nucleolar RNAs, four intergenic RNAs and nine RNA structures located within the introns and untranslated regions (UTRs) of mRNAs. For the two structures in the 3' UTRs of the metabolic genes GLY1 and MET13, we performed experiments that provide evidence against them being eukaryotic riboswitches.
Asunto(s)
ARN de Hongos/química , ARN no Traducido/química , Regiones no Traducidas 3' , Biología Computacional/métodos , Genoma Fúngico , Intrones , Lisina-ARNt Ligasa/genética , Cadenas de Markov , Conformación de Ácido Nucleico , ARN Nucleolar Pequeño/química , Proteínas Ribosómicas/genética , Riboswitch , Alineación de Secuencia , Tiorredoxinas/genéticaRESUMEN
RNA exosomopathies, a growing family of diseases, are linked to missense mutations in genes encoding structural subunits of the evolutionarily conserved, 10-subunit exoribonuclease complex, the RNA exosome. This complex consists of a three-subunit cap, a six-subunit, barrel-shaped core, and a catalytic base subunit. While a number of mutations in RNA exosome genes cause pontocerebellar hypoplasia, mutations in the cap subunit gene EXOSC2 cause an apparently distinct clinical presentation that has been defined as a novel syndrome SHRF (short stature, hearing loss, retinitis pigmentosa, and distinctive facies). We generated the first in vivo model of the SHRF pathogenic amino acid substitutions using budding yeast by modeling pathogenic EXOSC2 missense mutations (p.Gly30Val and p.Gly198Asp) in the orthologous S. cerevisiae gene RRP4 The resulting rrp4 mutant cells show defects in cell growth and RNA exosome function. Consistent with altered RNA exosome function, we detect significant transcriptomic changes in both coding and noncoding RNAs in rrp4-G226D cells that model EXOSC2 p.Gly198Asp, suggesting defects in nuclear surveillance. Biochemical and genetic analyses suggest that the Rrp4 G226D variant subunit shows impaired interactions with key RNA exosome cofactors that modulate the function of the complex. These results provide the first in vivo evidence that pathogenic missense mutations present in EXOSC2 impair the function of the RNA exosome. This study also sets the stage to compare exosomopathy models to understand how defects in RNA exosome function underlie distinct pathologies.
Asunto(s)
Exorribonucleasas/genética , Complejo Multienzimático de Ribonucleasas del Exosoma/genética , Mutación Missense , ARN de Hongos/genética , Proteínas de Unión al ARN/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Enanismo/enzimología , Enanismo/genética , Enanismo/patología , Exorribonucleasas/química , Exorribonucleasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/química , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Facies , Expresión Génica , Glicina/química , Glicina/metabolismo , Pérdida Auditiva/enzimología , Pérdida Auditiva/genética , Pérdida Auditiva/patología , Humanos , Modelos Biológicos , Modelos Moleculares , Conformación Proteica , ARN de Hongos/química , ARN de Hongos/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Retinitis Pigmentosa/enzimología , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/patología , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , SíndromeRESUMEN
By interacting with the mRNA 5' cap, the translation initiation factor eIF4E plays a critical role in selecting mRNAs for protein synthesis in eukaryotic cells. Caf20 is a member of the family of proteins found across eukaryotes termed 4E-BPs, which compete with eIF4G for interaction with eIF4E. Caf20 independently interacts with ribosomes. Thus, Caf20 modulates the mRNA selection process via poorly understood mechanisms. Here we performed unbiased mutagenesis across Caf20 to characterise which regions of Caf20 are important for interaction with eIF4E and with ribosomes. Caf20 binding to eIF4E is entirely dependent on a canonical motif shared with other 4E-BPs. However, binding to ribosomes is weakened by mutations throughout the protein, suggesting an extended binding interface that partially overlaps with the eIF4E-interaction region. By using chemical crosslinking, we identify a potential ribosome interaction region on the ribosome surface that spans both small and large subunits and is close to a known interaction site of eIF3. The function of ribosome binding by Caf20 remains unclear.
Asunto(s)
Factor 4E Eucariótico de Iniciación/química , ARN de Hongos/química , ARN Mensajero/química , Ribosomas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Factores de Transcripción/química , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Mutación , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Dicer is a member of the ribonuclease III enzyme family and processes double-stranded RNA into small functional RNAs. The variation in the domain architecture of Dicer among different species whilst preserving its biological dicing function is intriguing. Here, we describe the structure and function of a novel catalytically active RNase III protein, a non-canonical Dicer (PsDCR1), found in budding yeast Pichia stipitis. The structure of the catalytically active region (the catalytic RNase III domain and double-stranded RNA-binding domain 1 [dsRBD1]) of DCR1 showed that RNaseIII domain is structurally similar to yeast RNase III (Rnt1p) but uniquely presents dsRBD1 in a diagonal orientation, forming a catalytic core made of homodimer and large RNA-binding surface. The second dsRNA binding domain at C-terminus, which is absent in Rnt1, enhances the RNA cleavage activity. Although the cleavage pattern of PsDCR1 anchors an apical loop similar to Rnt1, the cleavage activity depended on the sequence motif at the lower stem, not the apical loop, of hairpin RNA. Through RNA sequencing and RNA mutations, we showed that RNA cleavage by PsDCR1 is determined by the stem-loop structure of the RNA substrate, suggesting the possibility that stem-loop RNA-guided gene silencing pathway exists in budding yeast.
Asunto(s)
Proteínas Fúngicas/química , Conformación de Ácido Nucleico , Multimerización de Proteína , ARN de Hongos/química , Ribonucleasa III/química , Saccharomycetales/enzimología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Dominios Proteicos , Estructura Secundaria de Proteína , ARN de Hongos/genética , ARN de Hongos/metabolismo , Ribonucleasa III/genética , Ribonucleasa III/metabolismo , Saccharomycetales/genética , Relación Estructura-ActividadRESUMEN
Precipitation is a critical step to recover RNA of high purity. This chapter describes the principles of alcoholic precipitation as well as a standard, basic protocol with key advices to observe, but numerous variations on the theme are discussed. Indeed, several important parameters, such as the choice of salt, alcohol, or carrier, have to be considered to improve the efficiency of precipitation and the yield of RNA recovery.
Asunto(s)
ARN de Transferencia/química , ARN de Transferencia/aislamiento & purificación , Levaduras/genética , Alcoholes/química , Precipitación Química , ARN de Hongos/química , Sales (Química)/químicaRESUMEN
Many RNA architectures were discovered to be involved in a wide range of essential biological processes in all organisms from carrying genetic information to gene expression regulation. The remarkable ability of RNAs to adopt various architectures depending on their environment enables the achievement of their myriads of biological functions. Nuclear Magnetic Resonance (NMR) is a powerful technique to investigate both their structure and dynamics. NMR is also a key tool for studying interactions between RNAs and their numerous partners such as small molecules, ions, proteins, or other nucleic acids.In this chapter, to illustrate the use of NMR for 3D structure determination of small noncoding RNA, we describe detailed methods that we used for the yeast C/D box small nucleolar RNA U14 from sample preparation to 3D structure calculation.
Asunto(s)
ARN Pequeño no Traducido/química , Saccharomyces cerevisiae/genética , Resonancia Magnética Nuclear Biomolecular , Conformación de Ácido Nucleico , ARN de Hongos/química , ARN de Hongos/metabolismo , ARN Pequeño no Traducido/metabolismoRESUMEN
The ATPase Prp16 governs equilibrium between the branching (B∗/C) and exon ligation (C∗/P) conformations of the spliceosome. Here, we present the electron cryomicroscopy reconstruction of the Saccharomyces cerevisiae C-complex spliceosome at 2.8 Å resolution and identify a novel C-complex intermediate (Ci) that elucidates the molecular basis for this equilibrium. The exon-ligation factors Prp18 and Slu7 bind to Ci before ATP hydrolysis by Prp16 can destabilize the branching conformation. Biochemical assays suggest that these pre-bound factors prime the C complex for conversion to C∗ by Prp16. A complete model of the Prp19 complex (NTC) reveals how the branching factors Yju2 and Isy1 are recruited by the NTC before branching. Prp16 remodels Yju2 binding after branching, allowing Yju2 to remain tethered to the NTC in the C∗ complex to promote exon ligation. Our results explain how Prp16 action modulates the dynamic binding of step-specific factors to alternatively stabilize the C or C∗ conformation and establish equilibrium of the catalytic spliceosome.
Asunto(s)
Modelos Químicos , Empalme del ARN , ARN de Hongos/química , Proteínas de Unión al ARN/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Empalmosomas/química , ARN de Hongos/genética , ARN de Hongos/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Empalmosomas/genética , Empalmosomas/metabolismoRESUMEN
Trichothecenes are the most prevalent mycotoxins contaminating cereal grains. Some of them are also considered as the virulence factors of Fusarium head blight disease. However, the mechanism behind the structure-activity relationship for trichothecenes remains unexplained. Filling this information gap is a crucial step for developing strategies to manage this large family of mycotoxins in food and feed. Here, we perform an in-depth re-examination of the existing structures of Saccharomyces cerevisiae ribosome complexed with three different trichothecenes. Multiple binding interactions between trichothecenes and 25S rRNA, including hydrogen bonds, nonpolar pi stacking interactions and metal ion coordination interactions, are identified as important binding determinants. These interactions are mainly contributed by the key structural elements to the toxicity of trichothecenes, including the oxygen in the 12,13-epoxide ring and a double bond between C9 and C10. In addition, the C3-OH group also participates in binding. The comparison of three trichothecenes binding to the ribosome, along with their binding pocket architecture, suggests that the substitutions at different positions impact trichothecenes binding in two different patterns. Moreover, the binding of trichothecenes induced conformation changes of several nucleotide bases in 25S rRNA. This then provides a structural framework for understanding the structure-activity relationships apparent in trichothecenes. This study will facilitate the development of strategies aimed at detoxifying mycotoxins in food and feed and at improving the resistance of cereal crops to Fusarium fungal diseases.
Asunto(s)
Micotoxinas/química , Tricotecenos/química , Sitios de Unión , Grano Comestible/toxicidad , Contaminación de Alimentos , Fusarium/química , Fusarium/patogenicidad , Inactivación Metabólica , Modelos Moleculares , Estructura Molecular , Micotoxinas/metabolismo , Micotoxinas/toxicidad , Conformación de Ácido Nucleico/efectos de los fármacos , ARN de Hongos/química , ARN de Hongos/efectos de los fármacos , ARN Ribosómico/química , ARN Ribosómico/efectos de los fármacos , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Relación Estructura-Actividad , Tricotecenos/metabolismo , Tricotecenos/toxicidadRESUMEN
3-(Methylthio)-1-propanol, reminiscent of cauliflower and cooked vegetable aroma, is an important sulfur compound in Baijiu. It is important to develop a method to increase 3-(methylthio)-1-propanol content to improve flavor quality of products. In this study, a synthetic microbial community was employed to enhance the content of 3-(methylthio)-1-propanol by multi-module division of labor approach. Firstly, the synthetic pathway of 3-(methylthio)-1-propanol was reconstructed and classified into three modules. Later, the hyper producers in each module were isolated and negative interaction between the members was relieved. Finally, a synthetic microbial community was constructed using three species containing one hyper producer from each module. Furthermore, the transcription characteristics of the species in each module were validated by metatranscriptomic analysis. The constructed synthetic microbial community can be used to biosynthesize 3-(methylthio)-1-propanol for Baijiu. This work provided a novel and workable strategy to design synthetic microbial community to enhance the flavor feature of other fermented foods.
Asunto(s)
Bacillus/metabolismo , Compuestos de Azufre/metabolismo , Bacillus/genética , Técnicas de Cultivo Celular por Lotes , Biomasa , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Lactobacillus/genética , Lactobacillus/metabolismo , Metionina/análisis , Metionina/metabolismo , ARN Bacteriano/química , ARN Bacteriano/aislamiento & purificación , ARN Bacteriano/metabolismo , ARN de Hongos/química , ARN de Hongos/aislamiento & purificación , ARN de Hongos/metabolismo , Saccharomyces/genética , Saccharomyces/metabolismo , Compuestos de Azufre/químicaRESUMEN
mRNA binding proteins regulate gene expression by controlling the processing, localization, decay, and translation of messenger RNAs (mRNAs). To fully understand these mechanisms of posttranscriptional gene regulation, it is necessary to identify the complete set of mRNA binding proteins. In recent years, several assays have been developed to accomplish this goal in a wide variety of organisms. This work describes a method for the systematic identification of mRNA binding proteins in Saccharomyces cerevisiae. This method applies in vivo UV cross-linking, affinity pull-down of polyA(+) mRNAs, and analysis by mass spectrometry to identify proteins that directly bind to mRNAs.
Asunto(s)
Espectrometría de Masas/métodos , ARN de Hongos/química , ARN Mensajero/química , Proteínas de Unión al ARN/química , Proteínas de Saccharomyces cerevisiae/química , Regulación Fúngica de la Expresión Génica , Unión Proteica , Saccharomyces cerevisiae/químicaRESUMEN
RNA helicases function in all aspects of RNA biology mainly through remodeling structures of RNA and RNA-protein (RNP) complexes. Among them, DEAD-box proteins form the largest family in eukaryotes and have been shown to remodel RNA/RNP structures and clamping of RNA-binding proteins, both in vitro and in vivo. Nevertheless, for the majority of these enzymes, it is largely unclear what RNAs are targeted and where they modulate RNA/RNP structures to promote RNA metabolism. Several methods have been developed to probe secondary and tertiary structures of specific transcripts or whole transcriptomes in vivo. In this chapter, we describe a protocol for identification of RNA structural changes that are dependent on a Saccharomyces cerevisiae DEAD-box helicase Dbp2. Experiments detailed here can be adapted to the study of other RNA helicases and identification of putative remodeling targets in vivo.