RESUMEN
mRNA biogenesis in the eukaryotic nucleus is a highly complex process. The numerous RNA processing steps are tightly coordinated to ensure that only fully processed transcripts are released from chromatin for export from the nucleus. Here, we present the hypothesis that fission yeast Dbp2, a ribonucleoprotein complex (RNP) remodelling ATPase of the DEAD-box family, is the key enzyme in an RNP assembly checkpoint at the 3'-end of genes. We show that Dbp2 interacts with the cleavage and polyadenylation complex (CPAC) and localises to cleavage bodies, which are enriched for 3'-end processing factors and proteins involved in nuclear RNA surveillance. Upon loss of Dbp2, 3'-processed, polyadenylated RNAs accumulate on chromatin and in cleavage bodies, and CPAC components are depleted from the soluble pool. Under these conditions, cells display an increased likelihood to skip polyadenylation sites and a delayed transcription termination, suggesting that levels of free CPAC components are insufficient to maintain normal levels of 3'-end processing. Our data support a model in which Dbp2 is the active component of an mRNP remodelling checkpoint that licenses RNA export and is coupled to CPAC release.
Asunto(s)
ARN Helicasas DEAD-box , Ribonucleoproteínas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Poliadenilación , ARN Mensajero/metabolismo , ARN Mensajero/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Cromatina/metabolismo , ARN de Hongos/metabolismo , ARN de Hongos/genética , Núcleo Celular/metabolismoRESUMEN
The 18S rRNA sequence is highly conserved, particularly at its 3'-end, which is formed by the endonuclease Nob1. How Nob1 identifies its target sequence is not known, and in vitro experiments have shown Nob1 to be error-prone. Moreover, the sequence around the 3'-end is degenerate with similar sites nearby. Here, we used yeast genetics, biochemistry, and next-generation sequencing to investigate a role for the ATPase Rio1 in monitoring the accuracy of the 18S rRNA 3'-end. We demonstrate that Nob1 can miscleave its rRNA substrate and that miscleaved rRNA accumulates upon bypassing the Rio1-mediated quality control (QC) step, but not in healthy cells with intact QC mechanisms. Mechanistically, we show that Rio1 binding to miscleaved rRNA is weaker than its binding to accurately processed 18S rRNA. Accordingly, excess Rio1 results in accumulation of miscleaved rRNA. Ribosomes containing miscleaved rRNA can translate, albeit more slowly, thereby inviting collisions with trailing ribosomes. These collisions result in degradation of the defective ribosomes utilizing parts of the machinery for mRNA QC. Altogether, the data support a model in which Rio1 inspects the 3'-end of the nascent 18S rRNA to prevent miscleaved 18S rRNA-containing ribosomes from erroneously engaging in translation, where they induce ribosome collisions. The data also demonstrate how ribosome collisions purify cells of altered ribosomes with different functionalities, with important implications for the concept of ribosome heterogeneity.
Asunto(s)
ARN Ribosómico 18S , Ribosomas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Ribosomas/metabolismo , División del ARN , Estabilidad del ARN/genética , ARN de Hongos/metabolismo , ARN de Hongos/genética , ARN Ribosómico 18S/metabolismo , ARN Ribosómico 18S/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Although lack of ADAR (adenosine deaminase acting on RNA) orthologs, genome-wide A-to-I editing occurs specifically during sexual reproduction in a number of filamentous ascomycetes, including Fusarium graminearum and Neurospora crassa. Unlike ADAR-mediated editing in animals, fungal A-to-I editing has a strong preference for hairpin loops and U at -1 position, which leads to frequent editing of UAG and UAA stop codons. Majority of RNA editing events in fungi are in the coding region and cause amino acid changes. Some of these editing events have been experimentally characterized for providing heterozygote and adaptive advantages in F. graminearum. Recent studies showed that FgTad2 and FgTad3, 2 ADAT (adenosine deaminase acting on tRNA) enzymes that normally catalyze the editing of A34 in the anticodon of tRNA during vegetative growth mediate A-to-I mRNA editing during sexual reproduction. Stage specificity of RNA editing is conferred by stage-specific expression of short transcript isoforms of FgTAD2 and FgTAD3 as well as cofactors such as AME1 and FIP5 that facilitate the editing of mRNA in perithecia. Taken together, fungal A-to-I RNA editing during sexual reproduction is catalyzed by ADATs and it has the same sequence and structural preferences with editing of A34 in tRNA.
Asunto(s)
Adenosina Desaminasa , Edición de ARN , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ascomicetos/genética , ARN de Hongos/genética , ARN de Hongos/metabolismo , Adenosina/metabolismo , Adenosina/genética , Inosina/metabolismo , Inosina/genética , Fusarium/genética , Neurospora crassa/genéticaRESUMEN
Factor-dependent termination uses molecular motors to remodel transcription machineries, but the associated mechanisms, especially in eukaryotes, are poorly understood. Here we use single-molecule fluorescence assays to characterize in real time the composition and the catalytic states of Saccharomyces cerevisiae transcription termination complexes remodeled by Sen1 helicase. We confirm that Sen1 takes the RNA transcript as its substrate and translocates along it by hydrolyzing multiple ATPs to form an intermediate with a stalled RNA polymerase II (Pol II) transcription elongation complex (TEC). We show that this intermediate dissociates upon hydrolysis of a single ATP leading to dissociation of Sen1 and RNA, after which Sen1 remains bound to the RNA. We find that Pol II ends up in a variety of states: dissociating from the DNA substrate, which is facilitated by transcription bubble rewinding, being retained to the DNA substrate, or diffusing along the DNA substrate. Our results provide a complete quantitative framework for understanding the mechanism of Sen1-dependent transcription termination in eukaryotes.
Asunto(s)
Adenosina Trifosfato , ADN Helicasas , ARN Polimerasa II , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Imagen Individual de Molécula , Terminación de la Transcripción Genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ARN Polimerasa II/metabolismo , Adenosina Trifosfato/metabolismo , ADN Helicasas/metabolismo , ADN Helicasas/genética , Imagen Individual de Molécula/métodos , ARN Helicasas/metabolismo , ARN Helicasas/genética , Transcripción Genética , ARN de Hongos/metabolismo , ARN de Hongos/genética , ADN de Hongos/metabolismo , ADN de Hongos/genética , HidrólisisRESUMEN
In eukaryotes, the Suv39 family of proteins tri-methylate lysine 9 of histone H3 (H3K9me) to form constitutive heterochromatin. However, how Suv39 proteins are nucleated at heterochromatin is not fully described. In the fission yeast, current models posit that Argonaute1-associated small RNAs (sRNAs) nucleate the sole H3K9 methyltransferase, Clr4/SUV39H, to centromeres. Here, we show that in the absence of all sRNAs and H3K9me, the Mtl1 and Red1 core (MTREC)/PAXT complex nucleates Clr4/SUV39H at a heterochromatic long noncoding RNA (lncRNA) at which the two H3K9 deacetylases, Sir2 and Clr3, also accumulate by distinct mechanisms. Iterative cycles of H3K9 deacetylation and methylation spread Clr4/SUV39H from the nucleation center in an sRNA-independent manner, generating a basal H3K9me state. This is acted upon by the RNAi machinery to augment and amplify the Clr4/H3K9me signal at centromeres to establish heterochromatin. Overall, our data reveal that lncRNAs and RNA quality control factors can nucleate heterochromatin and function as epigenetic silencers in eukaryotes.
Asunto(s)
Proteínas de Ciclo Celular , Heterocromatina , N-Metiltransferasa de Histona-Lisina , Histonas , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas de Ciclo Celular/metabolismo , Centrómero/metabolismo , Heterocromatina/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Metilación , Metiltransferasas/metabolismo , ARN Largo no Codificante/metabolismo , ARN Largo no Codificante/genética , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , ARN de Hongos/genética , ARN Interferente Pequeño/genéticaRESUMEN
Ribosomal RNAs (rRNAs) are extensively modified during the transcription and subsequent maturation. Three types of modifications, 2'-O-methylation of ribose moiety, pseudouridylation, and base modifications, are introduced either by a snoRNA-driven mechanism or by stand-alone enzymes. Modified nucleotides are clustered at the functionally important sites, including peptidyl transferase center (PTC). Therefore, it has been hypothesised that the modified nucleotides play an important role in ensuring the functionality of the ribosome. In this study, we demonstrate that seven 25S rRNA modifications, including four evolutionarily conserved modifications, in the proximity of PTC can be simultaneously depleted without loss of cell viability. Yeast mutants lacking three snoRNA genes (snR34, snR52, and snR65) and/or expressing enzymatically inactive variants of spb1(D52A/E679K) and nop2(C424A/C478A) were constructed. The results show that rRNA modifications in PTC contribute collectively to efficient translation in eukaryotic cells. The deficiency of seven modified nucleotides in 25S rRNA resulted in reduced cell growth, cold sensitivity, decreased translation levels, and hyperaccurate translation, as indicated by the reduced missense and nonsense suppression. The modification m5C2870 is crucial in the absence of the other six modified nucleotides. Thus, the pattern of rRNA-modified nucleotides around the PTC is essential for optimal ribosomal translational activity and translational fidelity.
Asunto(s)
Peptidil Transferasas , Biosíntesis de Proteínas , ARN Ribosómico , Saccharomyces cerevisiae , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Peptidil Transferasas/metabolismo , Peptidil Transferasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Ribosomas/metabolismo , ARN Nucleolar Pequeño/genética , ARN Nucleolar Pequeño/metabolismo , Procesamiento Postranscripcional del ARN , ARN de Hongos/genética , ARN de Hongos/metabolismo , MutaciónRESUMEN
Long noncoding RNAs (lncRNAs) are regulatory RNAs. Saccharomyces cerevisiae strains transcribe hundreds of lncRNAs. LncRNAs can regulate the expression of adjacent genes (cis-regulation) or distant genes from lncRNAs (trans-regulation). Here, we analyzed the potential global cis and trans-regulation of lncRNAs of yeast subjected to ethanol stress. For potential cis regulation, for BMA641-A and S288C strains, we observed that most lncRNA-neighbor gene pairs increased the expression at a certain point followed by a decrease, and vice versa. Based on the transcriptome profile and triple helix prediction between lncRNAs and promoters of coding genes, we observed nine different ways of potential trans regulation that work in a strain-specific manner. Our data provide an initial landscape of potential cis and trans regulation in yeast, which seems to be strain-specific.
Asunto(s)
Etanol , Regulación Fúngica de la Expresión Génica , ARN Largo no Codificante , Saccharomyces cerevisiae , Estrés Fisiológico , Saccharomyces cerevisiae/genética , ARN Largo no Codificante/genética , Etanol/farmacología , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Estrés Fisiológico/genética , Regiones Promotoras Genéticas , ARN de Hongos/genética , ARN de Hongos/metabolismo , Perfilación de la Expresión Génica/métodos , TranscriptomaRESUMEN
Ribosome biogenesis, involving processing/assembly of rRNAs and r-proteins is a vital process. In Saccharomyces cerevisiae mitochondria, ribosomal small subunit comprises 15S rRNA (15S). While the 15S 5'-end processing uses Ccm1p and Pet127p, the mechanisms of the 3'-end processing remain unclear. We reveal involvement of Rmd9p in safeguarding/processing 15S 3'-end. Rmd9p deficiency results in a cleavage at a position 183 nucleotides upstream of 15S 3'-end, and in the loss of the 3'-minor domain. Rmd9p binds to the sequences in the 3'-end region of 15S, and a genetic interaction between rmd9 and dss1 indicates that Rmd9p regulates/limits mtEXO activity during the 3'-end spacer processing.
Asunto(s)
ARN Ribosómico , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Mitocondrias/metabolismo , Mitocondrias/genética , Procesamiento de Término de ARN 3' , Procesamiento Postranscripcional del ARN , ARN de Hongos/metabolismo , ARN de Hongos/genética , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Systemic fungal infections are a growing public health threat, and yet viable antifungal drug targets are limited as fungi share a similar proteome with humans. However, features of RNA metabolism and the noncoding transcriptomes in fungi are distinctive. For example, fungi harbor highly structured RNA elements that humans lack, such as self-splicing introns within key housekeeping genes in the mitochondria. However, the location and function of these mitochondrial riboregulatory elements has largely eluded characterization. Here we used an RNA-structure-based bioinformatics pipeline to identify the group I introns interrupting key mitochondrial genes in medically relevant fungi, revealing their fixation within a handful of genetic hotspots and their ubiquitous presence across divergent phylogenies of fungi, including all highest priority pathogens such as Candida albicans, Candida auris, Aspergillus fumigatus and Cryptococcus neoformans. We then biochemically characterized two representative introns from C. albicans and C. auris, demonstrating their exceptionally efficient splicing catalysis relative to previously-characterized group I introns. Indeed, the C. albicans mitochondrial intron displays extremely rapid catalytic turnover, even at ambient temperatures and physiological magnesium ion concentrations. Our results unmask a significant new set of players in the RNA metabolism of pathogenic fungi, suggesting a promising new type of antifungal drug target.
Asunto(s)
Antifúngicos , Candida albicans , Intrones , Humanos , Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/genética , Intrones/genética , Empalme del ARN/genética , ARN de Hongos/metabolismoRESUMEN
Heat-shocked cells prioritize the translation of heat shock (HS) mRNAs, but the underlying mechanism is unclear. We report that HS in budding yeast induces the disassembly of the eIF4F complex, where eIF4G and eIF4E assemble into translationally arrested mRNA ribonucleoprotein particles (mRNPs) and HS granules (HSGs), whereas eIF4A promotes HS translation. Using in vitro reconstitution biochemistry, we show that a conformational rearrangement of the thermo-sensing eIF4A-binding domain of eIF4G dissociates eIF4A and promotes the assembly with mRNA into HS-mRNPs, which recruit additional translation factors, including Pab1p and eIF4E, to form multi-component condensates. Using extracts and cellular experiments, we demonstrate that HS-mRNPs and condensates repress the translation of associated mRNA and deplete translation factors that are required for housekeeping translation, whereas HS mRNAs can be efficiently translated by eIF4A. We conclude that the eIF4F complex is a thermo-sensing node that regulates translation during HS.
Asunto(s)
Factor 4F Eucariótico de Iniciación , Factor 4G Eucariótico de Iniciación , Respuesta al Choque Térmico , Proteínas de Unión a Poli(A) , Biosíntesis de Proteínas , ARN Mensajero , Ribonucleoproteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Respuesta al Choque Térmico/genética , Factor 4F Eucariótico de Iniciación/metabolismo , Factor 4F Eucariótico de Iniciación/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Factor 4A Eucariótico de Iniciación/genética , Regulación Fúngica de la Expresión Génica , Unión Proteica , ARN de Hongos/metabolismo , ARN de Hongos/genéticaRESUMEN
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
Small RNAs act as fungal pathogen effectors that silence host target genes to promote infection, a virulence mechanism termed cross-kingdom RNA interference (RNAi). The essential pathogen factors of cross-kingdom small RNA production are largely unknown. We here characterized the RNA-dependent RNA polymerase (RDR)1 in the fungal plant pathogen Botrytis cinerea that is required for pathogenicity and cross-kingdom RNAi. B. cinerea bcrdr1 knockout (ko) mutants exhibited reduced pathogenicity and loss of cross-kingdom small RNAs. We developed a "switch-on" GFP reporter to study cross-kingdom RNAi in real-time within the living plant tissue which highlighted that bcrdr1 ko mutants were compromised in cross-kingdom RNAi. Moreover, blocking seven pathogen cross-kingdom small RNAs by expressing a short-tandem target mimic RNA in transgenic Arabidopsis thaliana led to reduced infection levels of the fungal pathogen B. cinerea and the oomycete pathogen Hyaloperonospora arabidopsidis. These results demonstrate that cross-kingdom RNAi is significant to promote host infection and making pathogen small RNAs an effective target for crop protection.
Asunto(s)
Arabidopsis , ARN Polimerasa Dependiente del ARN , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Polimerasa Dependiente del ARN/genética , Arabidopsis/genética , Arabidopsis/microbiología , Virulencia/genética , Plantas/genética , Botrytis/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , ARN de Hongos/genética , ARN de PlantaRESUMEN
Despite the increasing number of fungal microRNA-like small RNAs (milRNAs) being identified and reported, profiling of milRNAs in biocontrol fungi and their roles in the mycoparasitism of pathogenic fungi remains limited. Therefore, in this study, we constructed a GFP fluorescence strain to evaluate the critical period of mycoparasitism in the interaction system between T. breve T069 and B. cinerea. The results showed that the early stage of Trichoderma mycoparasitism occurred 12 h after hyphal contact and was characterized by hyphal parallelism, whereas the middle stage lasted 36 h was characterized by wrapping. The late stage of mycoparasitism occurred at 72 h was characterized by the degradation of B. cinerea mycelia. We subsequently identified the sRNAs of T. breve T069 and B. cinerea during the critical period of mycoparasitism using high-throughput sequencing. In ltR1, 45 potential milRNA targets were identified for 243 genes, and 73 milRNAs targeted 733 genes in ltR3. Additionally, to identify potential transboundary miRNAs in T. breve T069, we screened for miRNAs that were exclusively expressed and had precursor structures in the T. breve T069 genome but were absent in the B. cinerea genome. Next, we predicted the target genes of B. cinerea. Our findings showed that 14 potential transboundary milRNAs from T. breve T069 targeted 41 genes in B. cinerea. Notably, cme-MIR164a-p5_1ss17CT can target 15 genes, including Rim15 (BCIN_15g00280), Nop53 (BCIN_12g03770), Skn7 (BCIN_02g08650), and Vel3 (BCIN_03g06410), while ppe-MIR477b-p3_1ss11TC targeted polyketide synthase (BCIN_03g04360, PKS3). The target gene of PC-5p-27397_41 was a non-ribosomal peptide synthetase (BCIN_01g03730, Bcnrps6). PC-3p-0029 (Tri-milR29) targeted chitin synthetase 7. These genes play crucial roles in normal mycelial growth and pathogenicity of B. cinerea. In conclusion, this study highlights the significance of milRNAs in Trichoderma mycoparasitism of B. cinerea. This discovery provides a new strategy for the application of miRNAs in the prevention and treatment of fungal pathogens.
Asunto(s)
Hypocreales , MicroARNs , Trichoderma , MicroARNs/genética , Hypocreales/genética , Botrytis/genética , ARN de Hongos/genética , Trichoderma/genética , Regulación Fúngica de la Expresión GénicaRESUMEN
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
Small RNAs (sRNAs) of the fungal pathogen Botrytis cinerea can enter plant cells and hijack host Argonaute protein 1 (AGO1) to silence host immunity genes. However, the mechanism by which these fungal sRNAs are secreted and enter host cells remains unclear. Here, we demonstrate that B. cinerea utilizes extracellular vesicles (EVs) to secrete Bc-sRNAs, which are then internalized by plant cells through clathrin-mediated endocytosis (CME). The B. cinerea tetraspanin protein, Punchless 1 (BcPLS1), serves as an EV biomarker and plays an essential role in fungal pathogenicity. We observe numerous Arabidopsis clathrin-coated vesicles (CCVs) around B. cinerea infection sites and the colocalization of B. cinerea EV marker BcPLS1 and Arabidopsis CLATHRIN LIGHT CHAIN 1, one of the core components of CCV. Meanwhile, BcPLS1 and the B. cinerea-secreted sRNAs are detected in purified CCVs after infection. Arabidopsis knockout mutants and inducible dominant-negative mutants of key components of the CME pathway exhibit increased resistance to B. cinerea infection. Furthermore, Bc-sRNA loading into Arabidopsis AGO1 and host target gene suppression are attenuated in those CME mutants. Together, our results demonstrate that fungi secrete sRNAs via EVs, which then enter host plant cells mainly through CME.
Asunto(s)
Arabidopsis , Vesículas Extracelulares , Arabidopsis/microbiología , ARN de Hongos/genética , Células Vegetales , Endocitosis , Clatrina , Enfermedades de las Plantas/microbiologíaRESUMEN
The establishment of host-microbe interactions requires molecular communication between both partners, which may involve the mutual transfer of noncoding small RNAs. Previous evidence suggests that this is also true for powdery mildew disease in barley, which is caused by the fungal pathogen Blumeria hordei. However, previous studies lacked spatial resolution regarding the accumulation of small RNAs upon host infection by B. hordei. Here, we analysed site-specific small RNA repertoires in the context of the barley-B. hordei interaction. To this end, we dissected infected leaves into separate fractions representing different sites that are key to the pathogenic process: epiphytic fungal mycelium, infected plant epidermis, isolated haustoria, a vesicle-enriched fraction from infected epidermis, and extracellular vesicles. Unexpectedly, we discovered enrichment of specific 31-33-base 5'-terminal fragments of barley 5.8S ribosomal RNA in extracellular vesicles and infected epidermis, as well as particular B. hordei transfer RNA fragments in haustoria. We describe canonical small RNAs from both the plant host and the fungal pathogen that may confer cross-kingdom RNA interference activity. Interestingly, we found first evidence of phased small interfering RNAs in B. hordei, a feature usually attributed to plants, which may be associated with the posttranscriptional control of fungal coding genes, pseudogenes, and transposable elements. Our data suggest a key and possibly site-specific role for cross-kingdom RNA interference and noncoding RNA fragments in the host-pathogen communication between B. hordei and its host barley.
Asunto(s)
Ascomicetos , Hordeum , ARN de Hongos/genética , Ascomicetos/genética , Ascomicetos/metabolismo , Hordeum/microbiología , ARN de Transferencia , Interferencia de ARN , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/metabolismoRESUMEN
Small RNAs (sRNAs) are short non-coding regulatory RNA sequences that silence the complementary expressive transcripts through an endogenous RNA mediated interference mechanism (RNAi). These sRNAs typically move through plasmodesmata and phloem in plants to support disease resistance, and also through septal pores and vesicles in fungi to act as effector of pathogenicity. Notably, recent reports have shown the occurrence of a bidirectional trafficking of these sRNAs between the host plants and the attacking fungal phytopathogen which have significant implication in the nature of the infection. While the trans-species sRNAs from the pathogen can silence the host mRNAs and inhibit the host immunity genes, the sRNA modules from the host plants can silence the mRNA in the pathogen by impeding the expression of the pathogenicity-related genes. In the present review, we discuss the current state of sRNA trafficking between the plant and the pathogen with special emphasis on the mechanism of cross-kingdom communication which could contribute to the development of pathogen and pest control in future agriculture.
Asunto(s)
Hongos , Plantas , ARN Pequeño no Traducido , Agricultura , Interferencia de ARN , ARN de Hongos/genética , ARN Mensajero , ARN Pequeño no Traducido/genética , Plantas/genética , Plantas/microbiología , Hongos/genética , Hongos/patogenicidadRESUMEN
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.
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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
miRNA-like RNAs (milRNAs) have been recognized as sequence-specific regulators of posttranscriptional regulation of gene expression in eukaryotes. However, the functions of hundreds of fungal milRNAs in the biosynthesis of metabolic components are obscure. Sanghuangporus produces diverse bioactive compounds and is widely used in Asian countries. Here, genes encoding two Dicers, four Argonautes, and four RdRPs were identified and characterized in Sanghuangporus vanini. Due to the lack of an efficient gene manipulation system, the efficacy of spray-induced gene silencing (SIGS) was determined in S. vanini, which showed efficient double-stranded RNA (dsRNA) uptake and gene silencing efficiency. SIGS-mediated gene knockdown showed that SVRDRP-3, SVRDRP-4, SVDICER-1, and SVDICER-2 were critical for mycelial biomass, flavonoid, triterpenoid, and polysaccharide production. Illumina deep sequencing was performed to characterize the milRNAs from S. vanini mycelium and fruiting body. A total of 31 milRNAs were identified, out of which, SvmilR10, SvmilR17, and SvmilR33 were Svrdrp-4- and Svdicer-1-dependent milRNAs. Importantly, SIGS-mediated overexpression of SvmilR10 and SvmilR33 resulted in significant changes in the yields of flavonoids, triterpenoids, and polysaccharides. Further analysis showed that these milRNA target genes encoding the retrotransposon-derived protein PEG1 and histone-lysine N-methyltransferase were potentially downregulated in the milRNA overexpressing strain. Our results revealed that S. vanini has high external dsRNA and small RNA uptake efficiency and that milRNAs may play crucial regulatory roles in the biosynthesis of bioactive compounds. IMPORTANCE Fungi can take up environmental RNA that can silence fungal genes with RNA interference, which prompts the development of SIGS. Efficient dsRNA and milRNA uptake in S. vanini, successful dsRNA-targeted gene block, and the increase in intracellular miRNA abundance showed that SIGS technology is an effective and powerful tool for the functional dissection of fungal genes and millRNAs. We found that the RdRP, Dicer, and Argonaute genes are critical for mycelial biomass and bioactive compound production. Our study also demonstrated that overexpressed SVRDRP-4- and SVDICER-1-dependent milRNAs (SvmilR10 and SvmilR33) led to significant changes in the yields of the three active compounds. This study not only provides the first report on SIGS-based gene and milRNA function exploration, but also provides a theoretical platform for exploration of the functions of milRNAs involved in biosynthesis of metabolic compounds in fungi.
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Basidiomycota , MicroARNs , MicroARNs/genética , MicroARNs/metabolismo , Basidiomycota/metabolismo , Interferencia de ARN , ARN de Hongos/genética , ARN de Hongos/metabolismo , Regulación Fúngica de la Expresión GénicaRESUMEN
Here, we report the characterization of a novel (-)ssRNA mycovirus isolated from Auricularia heimuer CCMJ1222, using a combination of RNA-seq, reverse transcription polymerase chain reaction, 5' and 3' rapid amplification of cDNA ends, and Sanger sequencing. Based on database searches, sequence alignment, and phylogenetic analysis, we designated the virus as "Auricularia heimuer negative-stranded RNA virus 1" (AhNsRV1). This virus has a monopartite RNA genome related to mymonaviruses (order Mononegavirales). The AhNsRV1 genome consists of 11,441 nucleotides and contains six open reading frames (ORFs). The largest ORF encodes a putative RNA-dependent RNA polymerase; the other ORFs encode hypothetical proteins with no conserved domains or known function. AhNsRV1 is the first (-)ssRNA virus and the third virus known to infect A. heimuer.