Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 8.175
Filtrar
1.
Nat Commun ; 11(1): 4104, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796835

RESUMO

Transfer RNAs (tRNA) are quintessential in deciphering the genetic code; disseminating nucleic acid triplets into correct amino acid identity. While this decoding function is clear, an emerging theme is that tRNA abundance and functionality can powerfully impact protein production rate, folding, activity, and messenger RNA stability. Importantly, however, the expression pattern of tRNAs is obliquely known. Here we present Quantitative Mature tRNA sequencing (QuantM-tRNA seq), a technique to monitor tRNA abundance and sequence variants secondary to RNA modifications. With QuantM-tRNA seq, we assess the tRNA transcriptome in mammalian tissues. We observe dramatic distinctions in isodecoder expression and known tRNA modifications between tissues. Remarkably, despite dramatic changes in tRNA isodecoder gene expression, the overall anticodon pool of each tRNA family is similar across tissues. These findings suggest that while anticodon pools appear to be buffered via an unknown mechanism, underlying transcriptomic and epitranscriptomic differences suggest a more complex tRNA regulatory landscape.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA de Transferência/metabolismo , Animais , Anticódon/genética , Northern Blotting , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , RNA Mensageiro/metabolismo , RNA de Transferência/genética
2.
Nat Commun ; 11(1): 4134, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807779

RESUMO

Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA degradation pathway that is important for the elimination of faulty, and the regulation of normal, mRNAs. The molecular details of the early steps in NMD are not fully understood but previous work suggests that NMD activation occurs as a consequence of ribosome stalling at the termination codon (TC). To test this hypothesis, we established an in vitro translation-coupled toeprinting assay based on lysates from human cells that allows monitoring of ribosome occupancy at the TC of reporter mRNAs. In contrast to the prevailing NMD model, our in vitro system reveals similar ribosomal occupancy at the stop codons of NMD-sensitive and NMD-insensitive reporter mRNAs. Moreover, ribosome profiling reveals a similar density of ribosomes at the TC of endogenous NMD-sensitive and NMD-insensitive mRNAs in vivo. Together, these data show that NMD activation is not accompanied by stable stalling of ribosomes at TCs.


Assuntos
Degradação do RNAm Mediada por Códon sem Sentido/fisiologia , Ribossomos/metabolismo , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/fisiologia , Códon de Terminação/genética , Humanos , Degradação do RNAm Mediada por Códon sem Sentido/genética , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética
3.
BMC Infect Dis ; 20(1): 585, 2020 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-32762666

RESUMO

BACKGROUND: The polymerase chain reaction (PCR) is commonly used to detect viral pathogens because of its high sensitivity and specificity. However, conventional PCR methods cannot determine virus infectivity. Virus infectivity is conventionally examined with methods such as the plaque assay, even though such assays require several days. Long-range reverse-transcription quantitative PCR (RT-qPCR) has previously been suggested for the rapid assessment of RNA virus infectivity where the loss of infectivity is attributable to genomic fragmentation. METHODS: IAV was irradiated with 253.7 nm ultraviolet (UV) rays to induce genomic strand breaks that were confirmed by a full-length RT-PCR assay. The IAV was then subjected to plaque assay, conventional RT-qPCR and long-range RT-qPCR to examine the relationship between infectious titer and copy number. A simple linear regression analysis was performed to examine the correlation between the results of these assays. RESULTS: A long-range RT-qPCR assay was developed and validated for influenza A virus (IAV). Although only a few minutes of UV irradiation was required to completely inactivate IAV, genomic RNA remained detectable by the conventional RT-qPCR and the full-length RT-PCR for NS of viral genome following inactivation. A long-range RT-qPCR assay was then designed using RT-priming at the 3' termini of each genomic segment and subsequent qPCR of the 5' regions. UV-mediated IAV inactivation was successfully analyzed by the long-range RT-qPCR assay especially when targeting PA of the viral genome. This was also supported by the regression analysis that the long-range RT-qPCR is highly correlated with plaque assay (Adjusted R2 = 0.931, P = 0.000066). CONCLUSIONS: This study suggests that IAV infectivity can be predicted without the infectivity assays. The rapid detection of pathogenic IAV has, therefore, been achieved with this sensing technology.


Assuntos
Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Infecções por Orthomyxoviridae/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Virulência/genética , Animais , Cães , Genoma Viral/genética , Genoma Viral/efeitos da radiação , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/efeitos da radiação , Células Madin Darby de Rim Canino , Infecções por Orthomyxoviridae/virologia , Estabilidade de RNA/efeitos da radiação , RNA Viral/genética , RNA Viral/efeitos da radiação , Raios Ultravioleta , Inativação de Vírus/efeitos da radiação
4.
PLoS One ; 15(8): e0237127, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32756602

RESUMO

BACKGROUND: The global pandemic of Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV2) has resulted in unprecedented challenges for healthcare systems. One barrier to widespread testing has been a paucity of traditional respiratory viral swab collection kits relative to the demand. Whether other sample collection kits, such as widely available MRSA nasal swabs can be used to detect SARS-CoV-2 is unknown. METHODS: We compared simultaneous nasal MRSA swabs (COPAN ESwabs ® 480C flocked nasal swab in 1mL of liquid Amies medium) and virals wabs (BD H192(07) flexible mini-tip flocked nasopharyngeal swabs in 3mL Universal Transport Medium) for SARS-CoV-2 PCR testing using Simplexa COVID-19 Direct assay on patients over a 4-day period. When the results were discordant, the viral swab sample was run again on the Cepheid Xpert Xpress ® SARS-CoV-2 assay. RESULTS: Of the 81 included samples, there were 19 positives and 62 negatives in viral media and 18 positives and 63 negative in the MRSA swabs. Amongst all included samples, there was concordance between the COPAN ESwabs ® 480C and the viral swabs in 78 (96.3%). CONCLUSION: We found a high rate of concordance in test results between COPAN ESwabs ® 480C in Amies solution and BD H192(07) nasopharyngeal swabs in in 3 mL of Universal Viral Transport medium viral media. Clinicians and laboratories should feel better informed and assured using COPAN ESwabs ® 480C to help in the diagnosis of COVID-19.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Staphylococcus aureus Resistente à Meticilina/genética , Pneumonia Viral/diagnóstico , Manejo de Espécimes/métodos , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Humanos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Nasofaringe/microbiologia , Nasofaringe/virologia , Pandemias , Pneumonia Viral/virologia , Estabilidade de RNA , RNA Bacteriano/análise , RNA Bacteriano/metabolismo , RNA Viral/análise , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
5.
Gene ; 759: 144988, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32717306

RESUMO

Hereditary familial adenomatous polyposis (FAP) in humans significantly increases the risk of development of colorectal cancer (CRC). Germline mutations in the APC (adenomatous polyposis coli) gene are responsible for FAP. Despite having the same causative mutation, the severity of the disease differs from patient to patient. The porcine FAP model carrying a truncating APC1311 mutation, orthologous to the dominant human mutation that leads to severe form of the disease (APC1309), mirrors the severity of polyposis. Earlier RNAseq studies have revealed the differential expression of WISP1 and CSF1R in samples derived from low-grade (LG-IEN) and more advanced high-grade (HG-IEN) colon polyps of APC1311/+ pigs. The grade of dysplasia was correlated with the severity of polyposis in APC1311/+ pigs characterized by a low (LP) and high (HP) numbers of polyps. The goal of this work was to find DNA variants that regulate the expression of CSF1R and WISP1 in LP and HP pigs. In total, 32 and 36 polymorphisms in CSF1R and WISP1 were found, respectively. Of these, the genotype frequency of four silent SNPs in the coding region of WISP1 differed significantly between LP and HP lines. In silico analysis revealed an elevated minimum free energy (MFE) for three of these SNPs, suggesting their role in mRNA structure stability. Furthermore, four polymorphisms in the promoter region of CSF1R, cosegregating as a common haplotype, were associated with polyp number in APC1311/+ pigs. A secreted alkaline phosphatase (SEAP) assay showed, however, that these variants have no direct effect on the activity of the CSF1R promoter. Concluding, our study identified polymorphisms in CSF1R and WISP1 that are potentially associated with the severity of polyposis in APC1311/+ pigs.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/genética , Proteínas de Sinalização Intercelular CCN/genética , Polimorfismo de Nucleotídeo Único , Receptores de Fator Estimulador de Colônias/genética , Polipose Adenomatosa do Colo/patologia , Animais , Proteínas de Sinalização Intercelular CCN/metabolismo , Modelos Animais de Doenças , Mutação , Estabilidade de RNA , Receptores de Fator Estimulador de Colônias/metabolismo , Suínos
6.
Wiley Interdiscip Rev RNA ; 11(5): e1614, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32638509

RESUMO

Coronaviruses, including SARS-Cov-2, are RNA-based pathogens that interface with a large variety of RNA-related cellular processes during infection. These processes include capping, polyadenylation, localization, RNA stability, translation, and regulation by RNA binding proteins or noncoding RNA effectors. The goal of this article is to provide an in-depth perspective on the current state of knowledge of how various coronaviruses interact with, usurp, and/or avoid aspects of these cellular RNA biology machineries. A thorough understanding of how coronaviruses interact with RNA-related posttranscriptional processes in the cell should allow for new insights into aspects of viral pathogenesis as well as identify new potential avenues for the development of anti-coronaviral therapeutics. This article is categorized under: RNA in Disease and Development > RNA in Disease.


Assuntos
Betacoronavirus/genética , Interações Hospedeiro-Patógeno/genética , MicroRNAs/genética , RNA Circular/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , RNA Viral/genética , Animais , Betacoronavirus/metabolismo , Humanos , MicroRNAs/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , Poliadenilação , Biossíntese de Proteínas , Edição de RNA , Processamento de RNA , Estabilidade de RNA , RNA Circular/metabolismo , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Vírus da SARS/genética , Vírus da SARS/metabolismo
7.
PLoS One ; 15(7): e0232559, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32658922

RESUMO

PRESENILIN 2 (PSEN2) is one of the genes mutated in early onset familial Alzheimer's disease (EOfAD). PSEN2 shares significant amino acid sequence identity with another EOfAD-related gene PRESENILIN 1 (PSEN1), and partial functional redundancy is seen between these two genes. However, the complete range of functions of PSEN1 and PSEN2 is not yet understood. In this study, we performed targeted mutagenesis of the zebrafish psen2 gene to generate a premature termination codon close downstream of the translation start with the intention of creating a null mutation. Homozygotes for this mutation, psen2S4Ter, are viable and fertile, and adults do not show any gross psen2-dependent pigmentation defects, arguing against significant loss of γ-secretase activity. Also, assessment of the numbers of Dorsal Longitudinal Ascending (DoLA) interneurons that are responsive to psen2 but not psen1 activity during embryogenesis did not reveal decreased psen2 function. Transcripts containing the S4Ter mutation show no evidence of destabilization by nonsense-mediated decay. Forced expression in zebrafish embryos of fusions of psen2S4Ter 5' mRNA sequences with sequence encoding enhanced green fluorescent protein (EGFP) indicated that the psen2S4Ter mutation permits utilization of cryptic, novel downstream translation start codons. These likely initiate translation of N-terminally truncated Psen2 proteins lacking late endosomal/lysosomal localization sequences and that obey the "reading frame preservation rule" of PRESENILIN EOfAD mutations. Transcriptome analysis of entire brains from a 6-month-old family of wild type, heterozygous and homozygous psen2S4Ter female siblings revealed profoundly dominant effects on gene expression likely indicating changes in ribosomal, mitochondrial, and anion transport functions.


Assuntos
Códon de Terminação/genética , Perfilação da Expressão Gênica , Mitocôndrias/genética , Mutação , Presenilina-2/genética , Ribossomos/genética , Proteínas de Peixe-Zebra/genética , Alelos , Animais , Contagem de Células , Homozigoto , Hipóxia/genética , Neurônios/citologia , Estabilidade de RNA/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética
8.
Nat Commun ; 11(1): 3717, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709887

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. 2'-O-RNA methyltransferase (MTase) is one of the enzymes of this virus that is a potential target for antiviral therapy as it is crucial for RNA cap formation; an essential process for viral RNA stability. This MTase function is associated with the nsp16 protein, which requires a cofactor, nsp10, for its proper activity. Here we show the crystal structure of the nsp10-nsp16 complex bound to the pan-MTase inhibitor sinefungin in the active site. Our structural comparisons reveal low conservation of the MTase catalytic site between Zika and SARS-CoV-2 viruses, but high conservation of the MTase active site between SARS-CoV-2 and SARS-CoV viruses; these data suggest that the preparation of MTase inhibitors targeting several coronaviruses - but not flaviviruses - should be feasible. Together, our data add to important information for structure-based drug discovery.


Assuntos
Betacoronavirus/enzimologia , Metiltransferases/química , Proteínas Virais Reguladoras e Acessórias/química , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Domínio Catalítico , Infecções por Coronavirus/virologia , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Metiltransferases/metabolismo , Modelos Químicos , Modelos Moleculares , Pandemias , Pneumonia Viral/virologia , Capuzes de RNA , Estabilidade de RNA , RNA Viral/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo
9.
Nat Commun ; 11(1): 3182, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576858

RESUMO

Most eukaryotic genes produce alternative polyadenylation (APA) isoforms. Here we report that, unlike previously characterized cell lineages, differentiation of syncytiotrophoblast (SCT), a cell type critical for hormone production and secretion during pregnancy, elicits widespread transcript shortening through APA in 3'UTRs and in introns. This global APA change is observed in multiple in vitro trophoblast differentiation models, and in single cells from placentas at different stages of pregnancy. Strikingly, the transcript shortening is unrelated to cell proliferation, a feature previously associated with APA control, but instead accompanies increased secretory functions. We show that 3'UTR shortening leads to transcripts with higher mRNA stability, which augments transcriptional activation, especially for genes involved in secretion. Moreover, this mechanism, named secretion-coupled APA (SCAP), is also executed in B cell differentiation to plasma cells. Together, our data indicate that SCAP tailors the transcriptome during formation of secretory cells, boosting their protein production and secretion capacity.


Assuntos
Diferenciação Celular/fisiologia , Poliadenilação/fisiologia , Transporte Proteico/fisiologia , Transcriptoma , Regiões 3' não Traduzidas , Diferenciação Celular/genética , Linhagem da Célula , Proliferação de Células , Células-Tronco Embrionárias , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Isoformas de Proteínas , Transporte Proteico/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo
10.
Viruses ; 12(6)2020 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-32486349

RESUMO

Porcine epidemic diarrhea virus (PEDV) causes a porcine disease associated with swine epidemic diarrhea. The type I interferon (IFN-I or IFN α/ß) is a key mediator of innate antiviral response during virus infection. Different antagonistic strategies have been identified and determined as to how PEDV infection inhibits the host's IFN responses to escape the host innate immune pathway, but the pathogenic mechanisms of PEDV infection are not fully elucidated. Our preliminary results revealed that endogenous TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3), the key components in the IFN signaling pathway were downregulated in PEDV infected IPEC-J2 cells by iTRAQ analysis. In this study, we screened nsp15 as the most important viral encoded protein involved in TBK1 and IRF3 reduction. Endoribonuclease (EndoU) activity has been well determined for coronavirus nsp15. Three residues (H226, H241, and K282) of PEDV nsp15 were identified as critical amino acids for PEDV EndoU but not D265, which was not well correlated with published results of other coronaviruses, such as severe acute respiratory syndrome virus (SARS-CoV). Moreover, PEDV nsp15 can directly degrade the RNA levels of TBK1 and IRF3 dependent on its EndoU activity to suppress IFN production and constrain the induction of IFN stimulated genes (ISGs), by which PEDV antagonizes the host innate response to facilitate its replication. Collectively, these results have confirmed that PEDV nsp15 was capable of subverting the IFN response by the RNA degradation of TBK1 and IRF3.


Assuntos
Endorribonucleases/imunologia , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/imunologia , Vírus da Diarreia Epidêmica Suína/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas não Estruturais Virais/imunologia , Animais , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Infecções por Coronavirus/veterinária , Regulação para Baixo , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/genética , Interferon Tipo I/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/imunologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/patologia , Células Vero
11.
Emerg Infect Dis ; 26(9)2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32511089

RESUMO

We found that environmental conditions affect the stability of severe acute respiratory syndrome coronavirus 2 in nasal mucus and sputum. The virus is more stable at low-temperature and low-humidity conditions, whereas warmer temperature and higher humidity shortened half-life. Although infectious virus was undetectable after 48 hours, viral RNA remained detectable for 7 days.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Muco/virologia , Pneumonia Viral/virologia , RNA Viral/análise , Escarro/virologia , Temperatura Alta , Humanos , Umidade , Cavidade Nasal/virologia , Pandemias , Estabilidade de RNA
12.
Proc Natl Acad Sci U S A ; 117(25): 14395-14404, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32513696

RESUMO

Retinoic acid-inducible gene I (RIG-I) is up-regulated during granulocytic differentiation of acute promyelocytic leukemia (APL) cells induced by all-trans retinoic acid (ATRA). It has been reported that RIG-I recognizes virus-specific 5'-ppp-double-stranded RNA (dsRNA) and activates the type I interferons signaling pathways in innate immunity. However, the functions of RIG-I in hematopoiesis remain unclear, especially regarding its possible interaction with endogenous RNAs and the associated pathways that could contribute to the cellular differentiation and maturation. Herein, we identified a number of RIG-I-binding endogenous RNAs in APL cells following ATRA treatment, including the tripartite motif-containing protein 25 (TRIM25) messenger RNA (mRNA). TRIM25 encodes the protein known as an E3 ligase for ubiquitin/interferon (IFN)-induced 15-kDa protein (ISG15) that is involved in RIG-I-mediated antiviral signaling. We show that RIG-I could bind TRIM25 mRNA via its helicase domain and C-terminal regulatory domain, enhancing the stability of TRIM25 transcripts. RIG-I could increase the transcriptional expression of TRIM25 by caspase recruitment domain (CARD) domain through an IFN-stimulated response element. In addition, RIG-I activated other key genes in the ISGylation pathway by activating signal transducer and activator of transcription 1 (STAT1), including the modifier ISG15 and several enzymes responsible for the conjugation of ISG15 to protein substrates. RIG-I cooperated with STAT1/2 and interferon regulatory factor 1 (IRF1) to promote the activation of the ISGylation pathway. The integrity of ISGylation in ATRA or RIG-I-induced cell differentiation was essential given that knockdown of TRIM25 or ISG15 resulted in significant inhibition of this process. Our results provide insight into the role of the RIG-I-TRIM25-ISGylation axis in myeloid differentiation.


Assuntos
Diferenciação Celular , Citocinas/metabolismo , Proteína DEAD-box 58/metabolismo , Granulócitos/fisiologia , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismo , Linhagem Celular Tumoral , Citocinas/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Estabilidade de RNA , RNA Mensageiro/metabolismo , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinas/genética , Regulação para Cima
13.
Proc Natl Acad Sci U S A ; 117(25): 14194-14201, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32522884

RESUMO

The intracellular environment is crowded and heterogeneous. Although the thermodynamic stability of nucleic acid duplexes is predictable in dilute solutions, methods of predicting such stability under specific intracellular conditions are not yet available. We recently showed that the nearest-neighbor model for self-complementary DNA is valid under molecular crowding condition of 40% polyethylene glycol with an average molecular weight of 200 (PEG 200) in 100 mM NaCl. Here, we determined nearest-neighbor parameters for DNA duplex formation under the same crowding condition to predict the thermodynamics of DNA duplexes in the intracellular environment. Preferential hydration of the nucleotides was found to be the key factor for nearest-neighbor parameters in the crowding condition. The determined parameters were shown to predict the thermodynamic parameters (∆H°, ∆S°, and ∆G°37) and melting temperatures (T m) of the DNA duplexes in the crowding condition with significant accuracy. Moreover, we proposed a general method for predicting the stability of short DNA duplexes in different cosolutes based on the relationship between duplex stability and the water activity of the cosolute solution. The method described herein would be valuable for investigating biological processes that occur under specific intracellular crowded conditions and for the application of DNA-based biotechnologies in crowded environments.


Assuntos
DNA/química , Nucleotídeos/química , Sequência de Bases , DNA/genética , Estrutura Molecular , Conformação de Ácido Nucleico , Polietilenoglicóis , RNA/química , Estabilidade de RNA , Termodinâmica
14.
Nat Commun ; 11(1): 3137, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561731

RESUMO

The close synergy between peptides and nucleic acids in current biology is suggestive of a functional co-evolution between the two polymers. Here we show that cationic proto-peptides (depsipeptides and polyesters), either produced as mixtures from plausibly prebiotic dry-down reactions or synthetically prepared in pure form, can engage in direct interactions with RNA resulting in mutual stabilization. Cationic proto-peptides significantly increase the thermal stability of folded RNA structures. In turn, RNA increases the lifetime of a depsipeptide by >30-fold. Proto-peptides containing the proteinaceous amino acids Lys, Arg, or His adjacent to backbone ester bonds generally promote RNA duplex thermal stability to a greater magnitude than do analogous sequences containing non-proteinaceous residues. Our findings support a model in which tightly-intertwined biological dependencies of RNA and protein reflect a long co-evolutionary history that began with rudimentary, mutually-stabilizing interactions at early stages of polypeptide and nucleic acid co-existence.


Assuntos
Evolução Molecular , Peptídeos/metabolismo , Dobramento de Proteína , Estabilidade de RNA , RNA/metabolismo , Sequência de Aminoácidos , Aminobutiratos/química , Aminobutiratos/metabolismo , Cátions/química , Cátions/metabolismo , Dicroísmo Circular , Hidrólise , Ressonância Magnética Nuclear Biomolecular , Origem da Vida , Ornitina/química , Ornitina/metabolismo , Peptídeos/química , Estabilidade Proteica , RNA/química , beta-Alanina/análogos & derivados , beta-Alanina/química , beta-Alanina/metabolismo
15.
Nat Commun ; 11(1): 3122, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561742

RESUMO

During nuclear surveillance in yeast, the RNA exosome functions together with the TRAMP complexes. These include the DEAH-box RNA helicase Mtr4 together with an RNA-binding protein (Air1 or Air2) and a poly(A) polymerase (Trf4 or Trf5). To better determine how RNA substrates are targeted, we analyzed protein and RNA interactions for TRAMP components. Mass spectrometry identified three distinct TRAMP complexes formed in vivo. These complexes preferentially assemble on different classes of transcripts. Unexpectedly, on many substrates, including pre-rRNAs and pre-mRNAs, binding specificity is apparently conferred by Trf4 and Trf5. Clustering of mRNAs by TRAMP association shows co-enrichment for mRNAs with functionally related products, supporting the significance of surveillance in regulating gene expression. We compared binding sites of TRAMP components with multiple nuclear RNA binding proteins, revealing preferential colocalization of subsets of factors. TRF5 deletion reduces Mtr4 recruitment and increases RNA abundance for mRNAs specifically showing high Trf5 binding.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Espectrometria de Massas , Mutação , Mapeamento de Interação de Proteínas , Precursores de RNA/metabolismo , Estabilidade de RNA , RNA-Seq , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato/genética
16.
Mol Cell ; 79(2): 251-267.e6, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32504555

RESUMO

The core components of the nuclear RNA export pathway are thought to be required for export of virtually all polyadenylated RNAs. Here, we depleted different proteins that act in nuclear export in human cells and quantified the transcriptome-wide consequences on RNA localization. Different genes exhibited substantially variable sensitivities, with depletion of NXF1 and TREX components causing some transcripts to become strongly retained in the nucleus while others were not affected. Specifically, NXF1 is preferentially required for export of single- or few-exon transcripts with long exons or high A/U content, whereas depletion of TREX complex components preferentially affects spliced and G/C-rich transcripts. Using massively parallel reporter assays, we identified short sequence elements that render transcripts dependent on NXF1 for their export and identified synergistic effects of splicing and NXF1. These results revise the current model of how nuclear export shapes the distribution of RNA within human cells.


Assuntos
Transporte Ativo do Núcleo Celular , Complexos Multiproteicos/metabolismo , Proteínas de Transporte Nucleocitoplasmático/fisiologia , Transporte de RNA , Proteínas de Ligação a RNA/fisiologia , RNA/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Núcleo Celular/metabolismo , Humanos , Camundongos , RNA/química , Estabilidade de RNA , RNA-Seq
17.
Nat Commun ; 11(1): 2823, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499480

RESUMO

FinO-domain proteins are a widespread family of bacterial RNA-binding proteins with regulatory functions. Their target spectrum ranges from a single RNA pair, in the case of plasmid-encoded FinO, to global RNA regulons, as with enterobacterial ProQ. To assess whether the FinO domain itself is intrinsically selective or promiscuous, we determine in vivo targets of Neisseria meningitidis, which consists of solely a FinO domain. UV-CLIP-seq identifies associations with 16 small non-coding sRNAs and 166 mRNAs. Meningococcal ProQ predominantly binds to highly structured regions and generally acts to stabilize its RNA targets. Loss of ProQ alters transcript levels of >250 genes, demonstrating that this minimal ProQ protein impacts gene expression globally. Phenotypic analyses indicate that ProQ promotes oxidative stress resistance and DNA damage repair. We conclude that FinO domain proteins recognize some abundant type of RNA shape and evolve RNA binding selectivity through acquisition of additional regions that constrain target recognition.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Neisseria meningitidis/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sequência de Bases , Dano ao DNA , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Neisseria meningitidis/genética , Conformação de Ácido Nucleico , Estresse Oxidativo , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Reprodutibilidade dos Testes , Relação Estrutura-Atividade
18.
Biochem Biophys Res Commun ; 527(4): 993-999, 2020 07 05.
Artigo em Inglês | MEDLINE | ID: covidwho-186318

RESUMO

Most viruses inhibit the innate immune system and/or the RNA degradation processes of host cells to construct an advantageous intracellular environment for their survival. Characteristic RNA sequences within RNA virus genomes or RNAs transcribed from DNA virus genomes contribute toward this inhibition. In this study, we developed a method called "Fate-seq" to comprehensively identify the RNA sequences derived from RNA and DNA viruses, contributing RNA stability in the cells. We examined the stabilization activity of 5,924 RNA fragments derived from 26 different viruses (16 RNA viruses and 10 DNA viruses) using next-generation sequencing of these RNAs fused 3' downstream of GFP reporter RNA. With the Fate-seq approach, we detected multiple virus-derived RNA sequences that stabilized GFP reporter RNA, including sequences derived from severe acute respiratory syndrome-related coronavirus (SARS-CoV). Comparative genomic analysis revealed that these RNA sequences and their predicted secondary structures are highly conserved between SARS-CoV and the novel coronavirus, SARS-CoV-2, which is responsible for the global outbreak of the coronavirus-associated disease that emerged in December 2019 (COVID-19). These sequences have the potential to enhance the stability of viral RNA genomes, thereby augmenting viral replication efficiency and virulence.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Estabilidade de RNA , RNA Viral/química , Vírus da SARS/genética , Sequência de Bases , Betacoronavirus/química , Sequência Conservada , Coronaviridae/genética , Genoma Viral , Células HeLa , Humanos , Conformação de Ácido Nucleico , Pandemias , Vírus da SARS/química , Análise de Sequência de RNA
19.
Nucleic Acids Res ; 48(12): 6919-6930, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32469055

RESUMO

Noncoding Y RNAs are abundant in animal cells and present in many bacteria. These RNAs are bound and stabilized by Ro60, a ring-shaped protein that is a target of autoantibodies in patients with systemic lupus erythematosus. Studies in bacteria revealed that Y RNA tethers Ro60 to a ring-shaped exoribonuclease, forming a double-ringed RNP machine specialized for structured RNA degradation. In addition to functioning as a tether, the bacterial RNA gates access of substrates to the Ro60 cavity. To identify roles for Y RNAs in mammals, we used CRISPR to generate mouse embryonic stem cells lacking one or both of the two murine Y RNAs. Despite reports that animal cell Y RNAs are essential for DNA replication, cells lacking these RNAs divide normally. However, Ro60 levels are reduced, revealing that Y RNA binding is required for Ro60 to accumulate to wild-type levels. Y RNAs regulate the subcellular location of Ro60, since Ro60 is reduced in the cytoplasm and increased in nucleoli when Y RNAs are absent. Last, we show that Y RNAs tether Ro60 to diverse effector proteins to generate specialized RNPs. Together, our data demonstrate that the roles of Y RNAs are intimately connected to that of their Ro60 partner.


Assuntos
Autoantígenos/genética , RNA Citoplasmático Pequeno/genética , RNA não Traduzido/genética , Ribonucleoproteínas/genética , Animais , Autoanticorpos/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Citoplasma/genética , Humanos , Camundongos , Conformação de Ácido Nucleico , Estabilidade de RNA/genética , RNA não Traduzido/ultraestrutura
20.
Nucleic Acids Res ; 48(11): 6265-6279, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32396167

RESUMO

P-bodies (PBs) are cytoplasmic mRNA-protein (mRNP) granules conserved throughout eukaryotes which are implicated in the repression, storage and degradation of mRNAs. PB assembly is driven by proteins with self-interacting and low-complexity domains. Non-translating mRNA also stimulates PB assembly, however no studies to date have explored whether particular mRNA transcripts are more critical than others in facilitating PB assembly. Previous work revealed that rps28bΔ (small ribosomal subunit-28B) mutants do not form PBs under normal growth conditions. Here, we demonstrate that the RPS28B 3'UTR is important for PB assembly, consistent with it harboring a binding site for the PB assembly protein Edc3. However, expression of the RPS28B 3'UTR alone is insufficient to drive PB assembly. Intriguingly, chimeric mRNA studies revealed that Rps28 protein, translated in cis from an mRNA bearing the RPS28B 3'UTR, physically interacts more strongly with Edc3 than Rps28 protein synthesized in trans. This Edc3-Rps28 interaction in turn facilitates PB assembly. Our work indicates that PB assembly may be nucleated by specific RNA 'scaffolds'. Furthermore, this is the first description in yeast to our knowledge of a cis-translated protein interacting with another protein in the 3'UTR of the mRNA which encoded it, which in turn stimulates assembly of cellular structures.


Assuntos
Estruturas Citoplasmáticas/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Regiões 3' não Traduzidas/genética , Deleção de Genes , Ligação Proteica , Estabilidade de RNA , Proteínas Ribossômicas/deficiência , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA