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1.
Nat Struct Mol Biol ; 28(9): 713-723, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34489609

RESUMO

Human mitochondrial transcripts contain messenger and ribosomal RNAs flanked by transfer RNAs (tRNAs), which are excised by mitochondrial RNase (mtRNase) P and Z to liberate all RNA species. In contrast to nuclear or bacterial RNase P, mtRNase P is not a ribozyme but comprises three protein subunits that carry out RNA cleavage and methylation by unknown mechanisms. Here, we present the cryo-EM structure of human mtRNase P bound to precursor tRNA, which reveals a unique mechanism of substrate recognition and processing. Subunits TRMT10C and SDR5C1 form a subcomplex that binds conserved mitochondrial tRNA elements, including the anticodon loop, and positions the tRNA for methylation. The endonuclease PRORP is recruited and activated through interactions with its PPR and nuclease domains to ensure precise pre-tRNA cleavage. The structure provides the molecular basis for the first step of RNA processing in human mitochondria.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/química , Metiltransferases/química , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Ribonuclease P/química , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Anticódon/química , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Microscopia Crioeletrônica , Humanos , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Mitocôndrias/enzimologia , Modelos Moleculares , Mutação de Sentido Incorreto , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , RNA Fúngico/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonuclease P/metabolismo , Especificidade da Espécie , Relação Estrutura-Atividade , Especificidade por Substrato
2.
Int J Mol Sci ; 22(16)2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34445460

RESUMO

Yeast phenotypes associated with the lack of wobble uridine (U34) modifications in tRNA were shown to be modulated by an allelic variation of SSD1, a gene encoding an mRNA-binding protein. We demonstrate that phenotypes caused by the loss of Deg1-dependent tRNA pseudouridylation are similarly affected by SSD1 allelic status. Temperature sensitivity and protein aggregation are elevated in deg1 mutants and further increased in the presence of the ssd1-d allele, which encodes a truncated form of Ssd1. In addition, chronological lifespan is reduced in a deg1 ssd1-d mutant, and the negative genetic interactions of the U34 modifier genes ELP3 and URM1 with DEG1 are aggravated by ssd1-d. A loss of function mutation in SSD1, ELP3, and DEG1 induces pleiotropic and overlapping phenotypes, including sensitivity against target of rapamycin (TOR) inhibitor drug and cell wall stress by calcofluor white. Additivity in ssd1 deg1 double mutant phenotypes suggests independent roles of Ssd1 and tRNA modifications in TOR signaling and cell wall integrity. However, other tRNA modification defects cause growth and drug sensitivity phenotypes, which are not further intensified in tandem with ssd1-d. Thus, we observed a modification-specific rather than general effect of SSD1 status on phenotypic variation in tRNA modification mutants. Our results highlight how the cellular consequences of tRNA modification loss can be influenced by protein targeting specific mRNAs.


Assuntos
Transferases Intramoleculares/deficiência , Processamento Pós-Transcricional do RNA/genética , RNA Fúngico , RNA de Transferência , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Variação Biológica da População , Transferases Intramoleculares/genética , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
3.
Nucleic Acids Res ; 49(15): 8535-8555, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34358317

RESUMO

Gene deletion and gene expression alteration can lead to growth defects that are amplified or reduced when a second mutation is present in the same cells. We performed 154 genetic interaction mapping (GIM) screens with query mutants related with RNA metabolism and estimated the growth rates of about 700 000 double mutant Saccharomyces cerevisiae strains. The tested targets included the gene deletion collection and 900 strains in which essential genes were affected by mRNA destabilization (DAmP). To analyze the results, we developed RECAP, a strategy that validates genetic interaction profiles by comparison with gene co-citation frequency, and identified links between 1471 genes and 117 biological processes. In addition to these large-scale results, we validated both enhancement and suppression of slow growth measured for specific RNA-related pathways. Thus, negative genetic interactions identified a role for the OCA inositol polyphosphate hydrolase complex in mRNA translation initiation. By analysis of suppressors, we found that Puf4, a Pumilio family RNA binding protein, inhibits ribosomal protein Rpl9 function, by acting on a conserved UGUAcauUA motif located downstream the stop codon of the RPL9B mRNA. Altogether, the results and their analysis should represent a useful resource for discovery of gene function in yeast.


Assuntos
Genes Fúngicos , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/genética , Alelos , Deleção de Genes , Pleiotropia Genética , Fosfatos de Inositol/metabolismo , Iniciação Traducional da Cadeia Peptídica , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia
4.
PLoS One ; 16(8): e0252365, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34351929

RESUMO

In filamentous fungi, gene silencing by RNA interference (RNAi) shapes many biological processes, including pathogenicity. Recently, fungal small RNAs (sRNAs) have been shown to act as effectors that disrupt gene activity in interacting plant hosts, thereby undermining their defence responses. We show here that the devastating mycotoxin-producing ascomycete Fusarium graminearum (Fg) utilizes DICER-like (DCL)-dependent sRNAs to target defence genes in two Poaceae hosts, barley (Hordeum vulgare, Hv) and Brachypodium distachyon (Bd). We identified 104 Fg-sRNAs with sequence homology to host genes that were repressed during interactions of Fg and Hv, while they accumulated in plants infected by the DCL double knock-out (dKO) mutant PH1-dcl1/2. The strength of target gene expression correlated with the abundance of the corresponding Fg-sRNA. Specifically, the abundance of three tRNA-derived fragments (tRFs) targeting immunity-related Ethylene overproducer 1-like 1 (HvEOL1) and three Poaceae orthologues of Arabidopsis thaliana BRI1-associated receptor kinase 1 (HvBAK1, HvSERK2 and BdSERK2) was dependent on fungal DCL. Additionally, RNA-ligase-mediated Rapid Amplification of cDNA Ends (RLM-RACE) identified infection-specific degradation products for the three barley gene transcripts, consistent with the possibility that tRFs contribute to fungal virulence via targeted gene silencing.


Assuntos
Brachypodium , Fusarium/fisiologia , Hordeum , Interações Hospedeiro-Patógeno , Doenças das Plantas/microbiologia , RNA Fúngico/metabolismo , Ribonuclease III/metabolismo , Fatores de Virulência/metabolismo , Brachypodium/metabolismo , Brachypodium/microbiologia , Proteínas Fúngicas , Hordeum/metabolismo , Hordeum/microbiologia , Doenças das Plantas/genética , RNA Fúngico/genética , Ribonuclease III/genética , Fatores de Virulência/genética
5.
Cells ; 10(8)2021 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-34440717

RESUMO

Recent findings suggest that ribosomes, the translational machineries, can display a distinct composition depending on physio-pathological contexts. Thanks to outstanding technological breakthroughs, many studies have reported that variations of rRNA modifications, and more particularly the most abundant rRNA chemical modification, the rRNA 2'O-ribose methylation (2'Ome), intrinsically occur in many organisms. In the last 5 years, accumulating reports have illustrated that rRNA 2'Ome varies in human cell lines but also in living organisms (yeast, plant, zebrafish, mouse, human) during development and diseases. These rRNA 2'Ome variations occur either within a single cell line, organ, or patient's sample (i.e., intra-variability) or between at least two biological conditions (i.e., inter-variability). Thus, the ribosomes can tolerate the absence of 2'Ome at some specific positions. These observations question whether variations in rRNA 2'Ome could provide ribosomes with particular translational regulatory activities and functional specializations. Here, we compile recent studies supporting the heterogeneity of ribosome composition at rRNA 2'Ome level and provide an overview of the natural diversity in rRNA 2'Ome that has been reported up to now throughout the kingdom of life. Moreover, we discuss the little evidence that suggests that variations of rRNA 2'Ome can effectively impact the ribosome activity and contribute to the etiology of some human diseases.


Assuntos
Evolução Molecular , Processamento Pós-Transcricional do RNA , RNA Fúngico/metabolismo , RNA Ribossômico/metabolismo , Ribossomos/metabolismo , Leveduras/metabolismo , Animais , Linhagem Celular , Humanos , Metilação , Biossíntese de Proteínas , RNA Fúngico/genética , RNA Ribossômico/genética , Ribossomos/genética , Leveduras/genética
6.
Cells ; 10(8)2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34440770

RESUMO

Introns are ubiquitous in eukaryotic genomes and have long been considered as 'junk RNA' but the huge energy expenditure in their transcription, removal, and degradation indicate that they may have functional significance and can offer evolutionary advantages. In fungi, plants and algae introns make a significant contribution to the size of the organellar genomes. Organellar introns are classified as catalytic self-splicing introns that can be categorized as either Group I or Group II introns. There are some biases, with Group I introns being more frequently encountered in fungal mitochondrial genomes, whereas among plants Group II introns dominate within the mitochondrial and chloroplast genomes. Organellar introns can encode a variety of proteins, such as maturases, homing endonucleases, reverse transcriptases, and, in some cases, ribosomal proteins, along with other novel open reading frames. Although organellar introns are viewed to be ribozymes, they do interact with various intron- or nuclear genome-encoded protein factors that assist in the intron RNA to fold into competent splicing structures, or facilitate the turn-over of intron RNAs to prevent reverse splicing. Organellar introns are also known to be involved in non-canonical splicing, such as backsplicing and trans-splicing which can result in novel splicing products or, in some instances, compensate for the fragmentation of genes by recombination events. In organellar genomes, Group I and II introns may exist in nested intronic arrangements, such as introns within introns, referred to as twintrons, where splicing of the external intron may be dependent on splicing of the internal intron. These nested or complex introns, with two or three-component intron modules, are being explored as platforms for alternative splicing and their possible function as molecular switches for modulating gene expression which could be potentially applied towards heterologous gene expression. This review explores recent findings on organellar Group I and II introns, focusing on splicing and mobility mechanisms aided by associated intron/nuclear encoded proteins and their potential roles in organellar gene expression and cross talk between nuclear and organellar genomes. Potential application for these types of elements in biotechnology are also discussed.


Assuntos
Íntrons , Organelas/genética , RNA de Algas/genética , RNA Fúngico/genética , RNA de Plantas/genética , RNA não Traduzido/genética , Evolução Molecular , Regulação Fúngica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genoma Fúngico , Genoma de Planta , Organelas/metabolismo , Splicing de RNA , Estabilidade de RNA , RNA de Algas/metabolismo , RNA Fúngico/metabolismo , RNA de Plantas/metabolismo , RNA não Traduzido/metabolismo , Transcrição Genética
7.
PLoS Comput Biol ; 17(8): e1008828, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34339411

RESUMO

Multinucleate cells occur in every biosphere and across the kingdoms of life, including in the human body as muscle cells and bone-forming cells. Data from filamentous fungi suggest that, even when bathed in a common cytoplasm, nuclei are capable of autonomous behaviors, including division. How does this potential for autonomy affect the organization of cellular processes between nuclei? Here we analyze a simplified model of circadian rhythm, a form of cellular oscillator, in a mathematical model of the filamentous fungus Neurospora crassa. Our results highlight a potential role played by mRNA-protein phase separation to keep mRNAs close to the nuclei from which they originate, while allowing proteins to diffuse freely between nuclei. Our modeling shows that syncytism allows for extreme mRNA efficiency-we demonstrate assembly of a robust oscillator with a transcription rate a thousand-fold less than in comparable uninucleate cells. We also show self-organized division of the labor of mRNA production, with one nucleus in a two-nucleus syncytium producing at least twice as many mRNAs as the other in 30% of cycles. This division can occur spontaneously, but division of labor can also be controlled by regulating the amount of cytoplasmic volume available to each nucleus. Taken together, our results show the intriguing richness and potential for emergent organization among nuclei in multinucleate cells. They also highlight the role of previously studied mechanisms of cellular organization, including nuclear space control and localization of mRNAs through RNA-protein phase separation, in regulating nuclear coordination.


Assuntos
Ritmo Circadiano/genética , Ritmo Circadiano/fisiologia , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Algoritmos , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Biologia Computacional , Simulação por Computador , Citoplasma/genética , Citoplasma/metabolismo , Células Gigantes/citologia , Células Gigantes/metabolismo , Humanos , Modelos Genéticos , Neurospora crassa/citologia , Neurospora crassa/genética , Neurospora crassa/fisiologia , RNA Fúngico/genética , RNA Fúngico/metabolismo , Processos Estocásticos , Transcrição Genética
8.
Nat Commun ; 12(1): 4231, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244499

RESUMO

Pathological aggregation of the protein tau into insoluble aggregates is a hallmark of neurodegenerative diseases. The emergence of disease-specific tau aggregate structures termed tau strains, however, remains elusive. Here we show that full-length tau protein can be aggregated in the absence of co-factors into seeding-competent amyloid fibrils that sequester RNA. Using a combination of solid-state NMR spectroscopy and biochemical experiments we demonstrate that the co-factor-free amyloid fibrils of tau have a rigid core that is similar in size and location to the rigid core of tau fibrils purified from the brain of patients with corticobasal degeneration. In addition, we demonstrate that the N-terminal 30 residues of tau are immobilized during fibril formation, in agreement with the presence of an N-terminal epitope that is specifically detected by antibodies in pathological tau. Experiments in vitro and in biosensor cells further established that co-factor-free tau fibrils efficiently seed tau aggregation, while binding studies with different RNAs show that the co-factor-free tau fibrils strongly sequester RNA. Taken together the study provides a critical advance to reveal the molecular factors that guide aggregation towards disease-specific tau strains.


Assuntos
Amiloide/metabolismo , Agregação Patológica de Proteínas/patologia , RNA/metabolismo , Proteínas tau/metabolismo , Amiloide/ultraestrutura , Técnicas Biossensoriais , Humanos , Ressonância Magnética Nuclear Biomolecular , RNA/ultraestrutura , RNA Fúngico/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas tau/isolamento & purificação , Proteínas tau/ultraestrutura
9.
Nat Commun ; 12(1): 4202, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244507

RESUMO

Biochemical reactions typically depend on the concentrations of the molecules involved, and cell survival therefore critically depends on the concentration of proteins. To maintain constant protein concentrations during cell growth, global mRNA and protein synthesis rates are tightly linked to cell volume. While such regulation is appropriate for most proteins, certain cellular structures do not scale with cell volume. The most striking example of this is the genomic DNA, which doubles during the cell cycle and increases with ploidy, but is independent of cell volume. Here, we show that the amount of histone proteins is coupled to the DNA content, even though mRNA and protein synthesis globally increase with cell volume. As a consequence, and in contrast to the global trend, histone concentrations decrease with cell volume but increase with ploidy. We find that this distinct coordination of histone homeostasis and genome content is already achieved at the transcript level, and is an intrinsic property of histone promoters that does not require direct feedback mechanisms. Mathematical modeling and histone promoter truncations reveal a simple and generalizable mechanism to control the cell volume- and ploidy-dependence of a given gene through the balance of the initiation and elongation rates.


Assuntos
Histonas/biossíntese , Modelos Genéticos , Biossíntese de Proteínas/genética , RNA Mensageiro/biossíntese , Transcrição Genética , DNA Fúngico/genética , Genoma Fúngico , Histonas/genética , Ploidias , Regiões Promotoras Genéticas/genética , RNA Fúngico/biossíntese , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética
10.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206573

RESUMO

Processing of the RNA polymerase I pre-rRNA transcript into the mature 18S, 5.8S, and 25S rRNAs requires removing the "spacer" sequences. The canonical pathway for the removal of the ITS1 spacer involves cleavages at the 3' end of 18S rRNA and at two sites inside ITS1. The process can generate either a long or a short 5.8S rRNA that differs in the number of ITS1 nucleotides retained at the 5.8S 5' end. Here we document a novel pathway to the long 5.8S, which bypasses cleavage within ITS1. Instead, the entire ITS1 is degraded from its 5' end by exonuclease Xrn1. Mutations in RNase MRP increase the accumulation of long relative to short 5.8S rRNA. Traditionally this is attributed to a decreased rate of RNase MRP cleavage at its target in ITS1, called A3. However, results from this work show that the MRP-induced switch between long and short 5.8S rRNA formation occurs even when the A3 site is deleted. Based on this and our published data, we propose that the link between RNase MRP and 5.8S 5' end formation involves RNase MRP cleavage at unknown sites elsewhere in pre-rRNA or in RNA molecules other than pre-rRNA.


Assuntos
RNA Ribossômico 5,8S/genética , RNA Ribossômico 5,8S/metabolismo , DNA Espaçador Ribossômico , Endorribonucleases , Regulação Fúngica da Expressão Gênica , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA , RNA Fúngico , RNA Ribossômico 5,8S/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Sequência
11.
Int J Mol Sci ; 22(14)2021 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-34299038

RESUMO

Ribosomal RNA is a major component of the ribosome. This RNA plays a crucial role in ribosome functioning by ensuring the formation of the peptide bond between amino acids and the accurate decoding of the genetic code. The rRNA carries many chemical modifications that participate in its maturation, the formation of the ribosome and its functioning. In this review, we present the different modifications and how they are deposited on the rRNA. We also describe the most recent results showing that the modified positions are not 100% modified, which creates a heterogeneous population of ribosomes. This gave rise to the concept of specialized ribosomes that we discuss. The knowledge accumulated in the yeast Saccharomyces cerevisiae is very helpful to better understand the role of rRNA modifications in humans, especially in ribosomopathies.


Assuntos
Modelos Biológicos , Processamento Pós-Transcricional do RNA , RNA Fúngico/genética , RNA Ribossômico/genética , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Humanos , Ribossomos/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
12.
Cells ; 10(6)2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200572

RESUMO

The implications of the microbiome on Coronavirus disease 2019 (COVID-19) prognosis has not been thoroughly studied. In this study we aimed to characterize the lung and blood microbiome and their implication on COVID-19 prognosis through analysis of peripheral blood mononuclear cell (PBMC) samples, lung biopsy samples, and bronchoalveolar lavage fluid (BALF) samples. In all three tissue types, we found panels of microbes differentially abundant between COVID-19 and normal samples correlated to immune dysregulation and upregulation of inflammatory pathways, including key cytokine pathways such as interleukin (IL)-2, 3, 5-10 and 23 signaling pathways and downregulation of anti-inflammatory pathways including IL-4 signaling. In the PBMC samples, six microbes were correlated with worse COVID-19 severity, and one microbe was correlated with improved COVID-19 severity. Collectively, our findings contribute to the understanding of the human microbiome and suggest interplay between our identified microbes and key inflammatory pathways which may be leveraged in the development of immune therapies for treating COVID-19 patients.


Assuntos
COVID-19/diagnóstico , Leucócitos Mononucleares/microbiologia , Pulmão/microbiologia , Microbiota/fisiologia , Líquido da Lavagem Broncoalveolar/microbiologia , Líquido da Lavagem Broncoalveolar/virologia , COVID-19/imunologia , COVID-19/microbiologia , COVID-19/virologia , Estudos de Casos e Controles , Humanos , Leucócitos Mononucleares/virologia , Biópsia Líquida , Pulmão/patologia , Pulmão/virologia , Microbiota/genética , Microbiota/imunologia , Prognóstico , RNA Bacteriano/análise , RNA Fúngico/análise , RNA-Seq , SARS-CoV-2/fisiologia
13.
Int J Mol Sci ; 22(13)2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34281276

RESUMO

Extracellular vesicles (EVs) are membranous, rounded vesicles released by prokaryotic and eukaryotic cells in their normal and pathophysiological states. These vesicles form a network of intercellular communication as they can transfer cell- and function-specific information (lipids, proteins and nucleic acids) to different cells and thus alter their function. Fungi are not an exception; they also release EVs to the extracellular space. The vesicles can also be retained in the periplasm as periplasmic vesicles (PVs) and the cell wall. Such fungal vesicles play various specific roles in the lives of these organisms. They are involved in creating wall architecture and maintaining its integrity, supporting cell isolation and defence against the environment. In the case of pathogenic strains, they might take part in the interactions with the host and affect the infection outcomes. The economic importance of fungi in manufacturing high-quality nutritional and pharmaceutical products and in remediation is considerable. The analysis of fungal EVs opens new horizons for diagnosing fungal infections and developing vaccines against mycoses and novel applications of nanotherapy and sensors in industrial processes.


Assuntos
Vesículas Extracelulares/fisiologia , Fungos/fisiologia , Transporte Biológico Ativo , Vesículas Extracelulares/genética , Vesículas Extracelulares/imunologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungos/genética , Fungos/patogenicidade , Genes Fúngicos , Interações entre Hospedeiro e Microrganismos/imunologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Modelos Biológicos , Micoses/diagnóstico , Micoses/microbiologia , RNA Fúngico/genética , RNA Fúngico/metabolismo
14.
Nat Commun ; 12(1): 4451, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294712

RESUMO

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.


Assuntos
DNA Fúngico/química , Estruturas R-Loop , RNA Fúngico/química , Ciclo Celular/genética , Ciclo Celular/fisiologia , Dano ao DNA , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , Genes Fúngicos , Instabilidade Genômica , Modelos Biológicos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estruturas R-Loop/genética , Estruturas R-Loop/fisiologia , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
15.
RNA ; 27(10): 1281-1290, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34272303

RESUMO

Gene expression in eukaryotes does not follow a linear process from transcription to translation and mRNA degradation. Instead it follows a circular process in which cytoplasmic mRNA decay crosstalks with nuclear transcription. In many instances, this crosstalk contributes to buffer mRNA at a roughly constant concentration. Whether the mRNA buffering concept operates on the total mRNA concentration or at the gene-specific level, and if the mechanism to do so is a global or a specific one, remain unknown. Here we assessed changes in mRNA concentrations and their synthesis rates along the transcriptome of aneuploid strains of the yeast Saccharomyces cerevisiae We also assessed mRNA concentrations and their synthesis rates in nonsense-mediated decay (NMD) targets in euploid strains. We found that the altered synthesis rates in the genes from the aneuploid chromosome and the changes in their mRNA stabilities were not counterbalanced. In addition, the stability of NMD targets was not specifically compensated by the changes in synthesis rate. We conclude that there is no genetic compensation of NMD mRNA targets in yeast, and total mRNA buffering uses mostly a global system rather than a gene-specific one.


Assuntos
Regulação Fúngica da Expressão Gênica , Genoma Fúngico , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Aneuploidia , Códon sem Sentido , Degradação do RNAm Mediada por Códon sem Sentido , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcriptoma
16.
PLoS Comput Biol ; 17(7): e1009188, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34297727

RESUMO

Cellular RNA levels typically fluctuate and are influenced by different transcription rates and RNA degradation rates. However, the understanding of the fundamental relationships between RNA abundance, environmental stimuli, RNA activities, and RNA age distributions is incomplete. Furthermore, the rates of RNA degradation and transcription are difficult to measure in transcriptomic experiments in living organisms, especially in studies involving humans. A model based on activity demands and RNA age was developed to explore the mechanisms of RNA level fluctuations. Using single-cell time-series gene expression experimental data, we assessed the transcription rates, RNA degradation rates, RNA life spans, RNA demand, accumulated transcription levels, and accumulated RNA degradation levels. This model could also predict RNA levels under simulation backgrounds, such as stimuli that induce regular oscillations in RNA abundance, stable RNA levels over time that result from long-term shortage of total RNA activity or from uncontrollable transcription, and relationships between RNA/protein levels and metabolic rates. This information contributes to existing knowledge.


Assuntos
Modelos Biológicos , Estabilidade de RNA , RNA Mensageiro/metabolismo , Biologia Computacional , Simulação por Computador , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Célula Única , Transcrição Genética , Transcriptoma
17.
PLoS One ; 16(7): e0254808, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34293017

RESUMO

MicroRNA-like small RNAs (milRNAs) and their regulatory roles in the interaction between plant and fungus have recently aroused keen interest of plant pathologists. Trichoderma spp., one of the widespread biocontrol fungi, can promote plant growth and induce plant disease resistance. To investigate milRNAs potentially involved in the interaction between Trichoderma and tomato roots, a small RNA (sRNA) library expressed during the interaction of T. asperellum DQ-1 and tomato roots was constructed and sequenced using the Illumina HiSeqTM 2500 sequencing platform. From 13,464,142 sRNA reads, we identified 21 milRNA candidates that were similar to other known microRNAs in the miRBase database and 22 novel milRNA candidates that possessed a stable microRNA precursor hairpin structure. Among them, three milRNA candidates showed different expression level in the interaction according to the result of stem-loop RT-PCR indicating that these milRNAs may play a distinct regulatory role in the interaction between Trichoderma and tomato roots. The potential transboundary milRNAs from T. asperellum and their target genes in tomato were predicted by bioinformatics analysis. The results revealed that several interesting proteins involved in plant growth and development, disease resistance, seed maturation, and osmotic stress signal transduction might be regulated by the transboundary milRNAs. To our knowledge, this is the first report of milRNAs taking part in the process of interaction of T. asperellum and tomato roots and associated with plant promotion and disease resistance. The results might be useful to unravel the mechanism of interaction between Trichoderma and tomato.


Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Hypocreales/fisiologia , Lycopersicon esculentum/microbiologia , MicroRNAs/biossíntese , Raízes de Plantas/microbiologia , RNA Fúngico/biossíntese , Resistência à Doença/fisiologia , MicroRNAs/genética , RNA Fúngico/genética
18.
BMC Infect Dis ; 21(1): 620, 2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34187390

RESUMO

BACKGROUND: Candida pelliculosa is an ecological fungal species that can cause infections in immunocompromised individuals. Numerous studies globally have shown that C. pelliculosa infects neonates. An outbreak recently occurred in our neonatal intensive care unit; therefore, we aimed to evaluate the risk factors in this hospital-acquired fungal infection. METHODS: We performed a case-control study, analysing the potential risk factors for neonatal infections of C. pelliculosa so that infection prevention and control could be implemented in our units. Isolated strains were tested for drug resistance and biofilm formation, important factors for fungal transmission that give rise to hospital-acquired infections. RESULTS: The use of three or more broad-spectrum antimicrobials or long hospital stays were associated with higher likelihoods of infection with C. pelliculosa. The fungus was not identified on the hands of healthcare workers or in the environment. All fungal isolates were susceptible to anti-fungal medications, and after anti-fungal treatment, all infected patients recovered. Strict infection prevention and control procedures efficiently suppressed infection transmission. Intact adhesin-encoding genes, shown by genome analysis, indicated possible routes for fungal transmission. CONCLUSIONS: The use of three or more broad-spectrum antimicrobials or a lengthy hospital stay is theoretically associated with the risk of infection with C. pelliculosa. Strains that we isolated are susceptible to anti-fungal medications, and these were eliminated by treating all patients with an antifungal. Transmission is likely via adhesion to the cell surface and biofilm formation.


Assuntos
Biofilmes , Candidíase/epidemiologia , Candidíase/prevenção & controle , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/prevenção & controle , Surtos de Doenças/prevenção & controle , Equipamentos e Provisões/microbiologia , Unidades de Terapia Intensiva Neonatal , Saccharomycetales/genética , Antifúngicos/uso terapêutico , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Estudos de Casos e Controles , China/epidemiologia , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/microbiologia , Feminino , Pessoal de Saúde , Humanos , Recém-Nascido , Controle de Infecções/métodos , Tempo de Internação , Masculino , Testes de Sensibilidade Microbiana , RNA Fúngico/genética , Fatores de Risco , Saccharomycetales/isolamento & purificação
19.
Nucleic Acids Res ; 49(11): 6128-6143, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34086938

RESUMO

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.


Assuntos
RNA Fúngico/química , RNA não Traduzido/química , Regiões 3' não Traduzidas , Biologia Computacional/métodos , Genoma Fúngico , Íntrons , Lisina-tRNA Ligase/genética , Cadeias de Markov , Conformação de Ácido Nucleico , RNA Nucleolar Pequeno/química , Proteínas Ribossômicas/genética , Riboswitch , Alinhamento de Sequência , Tiorredoxinas/genética
20.
RNA ; 27(9): 1046-1067, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34162742

RESUMO

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.


Assuntos
Exorribonucleases/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Mutação de Sentido Incorreto , RNA Fúngico/genética , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Nanismo/enzimologia , Nanismo/genética , Nanismo/patologia , Exorribonucleases/química , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/química , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Facies , Expressão Gênica , Glicina/química , Glicina/metabolismo , Perda Auditiva/enzimologia , Perda Auditiva/genética , Perda Auditiva/patologia , Humanos , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , RNA Fúngico/química , RNA Fúngico/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Retinite Pigmentosa/enzimologia , Retinite Pigmentosa/genética , Retinite Pigmentosa/patologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Síndrome
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