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1.
Mol Cell ; 80(1): 156-163.e6, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33007255

RESUMO

The production of alternative RNA variants contributes to the tissue-specific regulation of gene expression. In the animal nervous system, a systematic shift toward distal sites of transcription termination produces transcript signatures that are crucial for neuron development and function. Here, we report that, in Drosophila, the highly conserved protein ELAV globally regulates all sites of neuronal 3' end processing and directly binds to proximal polyadenylation sites of target mRNAs in vivo. We uncover an endogenous strategy of functional gene rescue that safeguards neuronal RNA signatures in an ELAV loss-of-function context. When not directly repressed by ELAV, the transcript encoding the ELAV paralog FNE acquires a mini-exon, generating a new protein able to translocate to the nucleus and rescue ELAV-mediated alternative polyadenylation and alternative splicing. We propose that exon-activated functional rescue is a more widespread mechanism that ensures robustness of processes regulated by a hierarchy, rather than redundancy, of effectors.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas ELAV/metabolismo , Éxons/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Masculino , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genética
2.
PLoS Pathog ; 16(8): e1008845, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866210

RESUMO

Modified vaccinia virus Ankara (MVA) is an approved smallpox vaccine and a promising vaccine vector for other pathogens as well as for cancer therapeutics with more than 200 current or completed clinical trials. MVA was derived by passaging the parental Ankara vaccine virus hundreds of times in chick embryo fibroblasts during which it lost the ability to replicate in human and most other mammalian cells. Although this replication deficiency is an important safety feature, the genetic basis of the host restriction is not understood. Here, an unbiased human genome-wide RNAi screen in human A549 cells revealed that the zinc-finger antiviral protein (ZAP), previously shown to inhibit certain RNA viruses, is a host restriction factor for MVA, a DNA virus. Additional studies demonstrated enhanced MVA replication in several human cell lines following knockdown of ZAP. Furthermore, CRISPR-Cas9 knockout of ZAP in human A549 cells increased MVA replication and spread by more than one log but had no effect on a non-attenuated strain of vaccinia virus. The intact viral C16 protein, which had been disrupted in MVA, antagonized ZAP by binding and sequestering the protein in cytoplasmic punctate structures. Studies aimed at exploring the mechanism by which ZAP restricts MVA replication in the absence of C16 showed that knockout of ZAP had no discernible effect on viral DNA or individual mRNA or protein species as determined by droplet digital polymerase chain reaction, deep RNA sequencing and mass spectrometry, respectively. Instead, inactivation of ZAP reduced the number of aberrant, dense, spherical particles that typically form in MVA-infected human cells, suggesting that ZAP has a novel role in interfering with a late step in the assembly of infectious MVA virions in the absence of the C16 protein.


Assuntos
Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Vírus Vaccinia/fisiologia , Replicação Viral/fisiologia , Células A549 , Animais , Galinhas , Citoplasma/metabolismo , Citoplasma/virologia , DNA Viral/genética , DNA Viral/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , RNA-Seq , Proteínas Repressoras/genética
3.
Nat Commun ; 11(1): 4625, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934225

RESUMO

A hallmark of neurodegeneration is defective protein quality control. The E3 ligase Listerin (LTN1/Ltn1) acts in a specialized protein quality control pathway-Ribosome-associated Quality Control (RQC)-by mediating proteolytic targeting of incomplete polypeptides produced by ribosome stalling, and Ltn1 mutation leads to neurodegeneration in mice. Whether neurodegeneration results from defective RQC and whether defective RQC contributes to human disease have remained unknown. Here we show that three independently-generated mouse models with mutations in a different component of the RQC complex, NEMF/Rqc2, develop progressive motor neuron degeneration. Equivalent mutations in yeast Rqc2 selectively interfere with its ability to modify aberrant translation products with C-terminal tails which assist with RQC-mediated protein degradation, suggesting a pathomechanism. Finally, we identify NEMF mutations expected to interfere with function in patients from seven families presenting juvenile neuromuscular disease. These uncover NEMF's role in translational homeostasis in the nervous system and implicate RQC dysfunction in causing neurodegeneration.


Assuntos
Doenças Neuromusculares/metabolismo , Ribossomos/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Doenças Neuromusculares/genética , Doenças Neuromusculares/patologia , Proteólise , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência
4.
PLoS Pathog ; 16(9): e1008844, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886716

RESUMO

The genomes of RNA and small DNA viruses of vertebrates display significant suppression of CpG dinucleotide frequencies. Artificially increasing dinucleotide frequencies results in substantial attenuation of virus replication, suggesting that these compositional changes may facilitate recognition of non-self RNA sequences. Recently, the interferon inducible protein ZAP, was identified as the host factor responsible for sensing CpG in viral RNA, through direct binding and possibly downstream targeting for degradation. Using an arrayed interferon stimulated gene expression library screen, we identified ZAPS, and its associated factor TRIM25, as inhibitors of human cytomegalovirus (HCMV) replication. Exogenous expression of ZAPS and TRIM25 significantly reduced virus replication while knockdown resulted in increased virus replication. HCMV displays a strikingly heterogeneous pattern of CpG representation with specific suppression of CpG motifs within the IE1 major immediate early transcript which is absent in subsequently expressed genes. We demonstrated that suppression of CpG dinucleotides in the IE1 gene allows evasion of inhibitory effects of ZAP. We show that acute virus replication is mutually exclusive with high levels of cellular ZAP, potentially explaining the higher levels of CpG in viral genes expressed subsequent to IE1 due to the loss of pressure from ZAP in infected cells. Finally, we show that TRIM25 regulates alternative splicing between the ZAP short and long isoforms during HCMV infection and interferon induction, with knockdown of TRIM25 resulting in decreased ZAPS and corresponding increased ZAPL expression. These results demonstrate for the first time that ZAP is a potent host restriction factor against large DNA viruses and that HCMV evades ZAP detection through suppression of CpG dinucleotides within the major immediate early 1 transcript. Furthermore, TRIM25 is required for efficient upregulation of the interferon inducible short isoform of ZAP through regulation of alternative splicing.


Assuntos
Processamento Alternativo , Ilhas de CpG , Infecções por Citomegalovirus/metabolismo , Citomegalovirus/fisiologia , Regulação Viral da Expressão Gênica , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Replicação Viral , Linhagem Celular , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/patologia , Humanos , Proteínas Imediatamente Precoces , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
5.
PLoS Pathog ; 16(9): e1008842, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32898178

RESUMO

Signaling through retinoic acid inducible gene I (RIG-I) like receptors (RLRs) is tightly regulated, with activation occurring upon sensing of viral nucleic acids, and suppression mediated by negative regulators. Under homeostatic conditions aberrant activation of melanoma differentiation-associated protein-5 (MDA5) is prevented through editing of endogenous dsRNA by RNA editing enzyme Adenosine Deaminase Acting on RNA (ADAR1). In addition, ADAR1 is postulated to play pro-viral and antiviral roles during viral infections that are dependent or independent of RNA editing activity. Here, we investigated the importance of ADAR1 isoforms in modulating influenza A virus (IAV) replication and revealed the opposing roles for ADAR1 isoforms, with the nuclear p110 isoform restricting versus the cytoplasmic p150 isoform promoting IAV replication. Importantly, we demonstrate that p150 is critical for preventing sustained RIG-I signaling, as p150 deficient cells showed increased IFN-ß expression and apoptosis during IAV infection, independent of RNA editing activity. Taken together, the p150 isoform of ADAR1 is important for preventing sustained RIG-I induced IFN-ß expression and apoptosis during viral infection.


Assuntos
Adenosina Desaminase/metabolismo , Apoptose , Proteína DEAD-box 58/metabolismo , Vírus da Influenza A/fisiologia , Influenza Humana/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais , Replicação Viral , Células A549 , Adenosina Desaminase/genética , Proteína DEAD-box 58/genética , Células HEK293 , Humanos , Influenza Humana/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas de Ligação a RNA/genética
6.
Int J Mol Sci ; 21(19)2020 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-32993015

RESUMO

The outbreak of a novel coronavirus SARS-CoV-2 responsible for the COVID-19 pandemic has caused a worldwide public health emergency. Due to the constantly evolving nature of the coronaviruses, SARS-CoV-2-mediated alterations on post-transcriptional gene regulations across human tissues remain elusive. In this study, we analyzed publicly available genomic datasets to systematically dissect the crosstalk and dysregulation of the human post-transcriptional regulatory networks governed by RNA-binding proteins (RBPs) and micro-RNAs (miRs) due to SARS-CoV-2 infection. We uncovered that 13 out of 29 SARS-CoV-2-encoded proteins directly interacted with 51 human RBPs, of which the majority of them were abundantly expressed in gonadal tissues and immune cells. We further performed a functional analysis of differentially expressed genes in mock-treated versus SARS-CoV-2-infected lung cells that revealed enrichment for the immune response, cytokine-mediated signaling, and metabolism-associated genes. This study also characterized the alternative splicing events in SARS-CoV-2-infected cells compared to the control, demonstrating that skipped exons and mutually exclusive exons were the most abundant events that potentially contributed to differential outcomes in response to the viral infection. A motif enrichment analysis on the RNA genomic sequence of SARS-CoV-2 clearly revealed the enrichment for RBPs such as SRSFs, PCBPs, ELAVs, and HNRNPs, suggesting the sponging of RBPs by the SARS-CoV-2 genome. A similar analysis to study the interactions of miRs with SARS-CoV-2 revealed functionally important miRs that were highly expressed in immune cells, suggesting that these interactions may contribute to the progression of the viral infection and modulate the host immune response across other human tissues. Given the need to understand the interactions of SARS-CoV-2 with key post-transcriptional regulators in the human genome, this study provided a systematic computational analysis to dissect the role of dysregulated post-transcriptional regulatory networks controlled by RBPs and miRs across tissue types during a SARS-CoV-2 infection.


Assuntos
Betacoronavirus/genética , Betacoronavirus/metabolismo , Infecções por Coronavirus/virologia , Redes Reguladoras de Genes , MicroRNAs/genética , Pneumonia Viral/virologia , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Regulação da Expressão Gênica , Genoma Viral , Humanos , MicroRNAs/metabolismo , Pandemias , Mapas de Interação de Proteínas , Proteínas de Ligação a RNA/genética
7.
PLoS Pathog ; 16(9): e1008803, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32956403

RESUMO

The Dearing isolate of Mammalian orthoreovirus (T3D) is a prominent model of virus-host relationships and a candidate oncolytic virotherapy. Closely related laboratory strains of T3D, originating from the same ancestral T3D isolate, were recently found to exhibit significantly different oncolytic properties. Specifically, the T3DPL strain had faster replication kinetics in a panel of cancer cells and improved tumor regression in an in vivo melanoma model, relative to T3DTD. In this study, we discover that T3DPL and T3DTD also differentially activate host signalling pathways and downstream gene transcription. At equivalent infectious dose, T3DTD induces higher IRF3 phosphorylation and expression of type I IFNs and IFN-stimulated genes (ISGs) than T3DPL. Using mono-reassortants with intermediate replication kinetics and pharmacological inhibitors of reovirus replication, IFN responses were found to inversely correlate with kinetics of virus replication. In other words, slow-replicating T3D strains induce more IFN signalling than fast-replicating T3D strains. Paradoxically, during co-infections by T3DPL and T3DTD, there was still high IRF3 phosphorylation indicating a phenodominant effect by the slow-replicating T3DTD. Using silencing and knock-out of RIG-I to impede IFN, we found that IFN induction does not affect the first round of reovirus replication but does prevent cell-cell spread in a paracrine fashion. Accordingly, during co-infections, T3DPL continues to replicate robustly despite activation of IFN by T3DTD. Using gene expression analysis, we discovered that reovirus can also induce a subset of genes in a RIG-I and IFN-independent manner; these genes were induced more by T3DPL than T3DTD. Polymorphisms in reovirus σ3 viral protein were found to control activation of RIG-I/ IFN-independent genes. Altogether, the study reveals that single amino acid polymorphisms in reovirus genomes can have large impact on host gene expression, by both changing replication kinetics and by modifying viral protein activity, such that two closely related T3D strains can induce opposite cytokine landscapes.


Assuntos
Proteínas do Capsídeo/metabolismo , Interferons/metabolismo , Polimorfismo Genético , RNA de Cadeia Dupla/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores do Ácido Retinoico/metabolismo , Infecções por Reoviridae/virologia , Replicação Viral , Proteínas do Capsídeo/genética , Citocinas , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/metabolismo , Orthoreovirus de Mamíferos/fisiologia , RNA de Cadeia Dupla/genética , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Receptores do Ácido Retinoico/genética , Infecções por Reoviridae/genética , Infecções por Reoviridae/metabolismo , Transdução de Sinais
8.
Nat Commun ; 11(1): 4455, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901005

RESUMO

Dysregulated alternative splicing (AS) driving carcinogenetic mitosis remains poorly understood. Here, we demonstrate that cancer metastasis-associated antigen 1 (MTA1), a well-known oncogenic chromatin modifier, broadly interacts and co-expresses with RBPs across cancers, contributing to cancerous mitosis-related AS. Using developed fCLIP-seq technology, we show that MTA1 binds abundant transcripts, preferentially at splicing-responsible motifs, influencing the abundance and AS pattern of target transcripts. MTA1 regulates the mRNA level and guides the AS of a series of mitosis regulators. MTA1 deletion abrogated the dynamic AS switches of variants for ATRX and MYBL2 at mitotic stage, which are relevant to mitosis-related tumorigenesis. MTA1 dysfunction causes defective mitotic arrest, leads to aberrant chromosome segregation, and results in chromosomal instability (CIN), eventually contributing to tumorigenesis. Currently, little is known about the RNA splicing during mitosis; here, we uncover that MTA1 binds transcripts and orchestrates dynamic splicing of mitosis regulators in tumorigenesis.


Assuntos
Carcinogênese/genética , Carcinogênese/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Mitose/fisiologia , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Processamento Alternativo , Animais , Sítios de Ligação/genética , Montagem e Desmontagem da Cromatina/genética , Instabilidade Cromossômica , Feminino , Células HCT116 , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Mitose/genética , Neoplasias/genética , Neoplasias/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Transativadores/antagonistas & inibidores , Transativadores/genética
9.
Nat Commun ; 11(1): 4106, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796827

RESUMO

Alternative ribosome-rescue factor B (ArfB) rescues ribosomes stalled on non-stop mRNAs by releasing the nascent polypeptide from the peptidyl-tRNA. By rapid kinetics we show that ArfB selects ribosomes stalled on short truncated mRNAs, rather than on longer mRNAs mimicking pausing on rare codon clusters. In combination with cryo-electron microscopy we dissect the multistep rescue pathway of ArfB, which first binds to ribosomes very rapidly regardless of the mRNA length. The selectivity for shorter mRNAs arises from the subsequent slow engagement step, as it requires longer mRNA to shift to enable ArfB binding. Engagement results in specific interactions of the ArfB C-terminal domain with the mRNA entry channel, which activates peptidyl-tRNA hydrolysis by the N-terminal domain. These data reveal how protein dynamics translate into specificity of substrate recognition and provide insights into the action of a putative rescue factor in mitochondria.


Assuntos
Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Microscopia Crioeletrônica , RNA Mensageiro/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Ribossomos/ultraestrutura
10.
Nat Commun ; 11(1): 4090, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796831

RESUMO

hnRNPA2 is a human ribonucleoprotein (RNP) involved in RNA metabolism. It forms fibrils both under cellular stress and in mutated form in neurodegenerative conditions. Previous work established that the C-terminal low-complexity domain (LCD) of hnRNPA2 fibrillizes under stress, and missense mutations in this domain are found in the disease multisystem proteinopathy (MSP). However, little is known at the atomic level about the hnRNPA2 LCD structure that is involved in those processes and how disease mutations cause structural change. Here we present the cryo-electron microscopy (cryoEM) structure of the hnRNPA2 LCD fibril core and demonstrate its capability to form a reversible hydrogel in vitro containing amyloid-like fibrils. Whereas these fibrils, like pathogenic amyloid, are formed from protein chains stacked into ß-sheets by backbone hydrogen bonds, they display distinct structural differences: the chains are kinked, enabling non-covalent cross-linking of fibrils and disfavoring formation of pathogenic steric zippers. Both reversibility and energetic calculations suggest these fibrils are less stable than pathogenic amyloid. Moreover, the crystal structure of the disease-mutation-containing segment (D290V) of hnRNPA2 suggests that the replacement fundamentally alters the fibril structure to a more stable energetic state. These findings illuminate how molecular interactions promote protein fibril networks and how mutation can transform fibril structure from functional to a pathogenic form.


Assuntos
Amiloide/química , Amiloide/metabolismo , Microscopia Crioeletrônica/métodos , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/ultraestrutura , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/química , Humanos , Hidrogéis/química , Proteínas de Ligação a RNA/química
11.
Nat Commun ; 11(1): 3806, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732922

RESUMO

Most triple-negative breast cancer (TNBC) patients fail to respond to T cell-mediated immunotherapies. Unfortunately, the molecular determinants are still poorly understood. Breast cancer is the disease genetically linked to a deficiency in autophagy. Here, we show that autophagy defects in TNBC cells inhibit T cell-mediated tumour killing in vitro and in vivo. Mechanistically, we identify Tenascin-C as a candidate for autophagy deficiency-mediated immunosuppression, in which Tenascin-C is Lys63-ubiquitinated by Skp2, particularly at Lys942 and Lys1882, thus promoting its recognition by p62 and leading to its selective autophagic degradation. High Tenascin-C expression is associated with poor prognosis and inversely correlated with LC3B expression and CD8+ T cells in TNBC patients. More importantly, inhibition of Tenascin-C in autophagy-impaired TNBC cells sensitizes T cell-mediated tumour killing and improves antitumour effects of single anti-PD1/PDL1 therapy. Our results provide a potential strategy for targeting TNBC with the combination of Tenascin-C blockade and immune checkpoint inhibitors.


Assuntos
Autofagia/imunologia , Linfócitos T CD8-Positivos/imunologia , Tenascina/metabolismo , Neoplasias de Mama Triplo Negativas/imunologia , Evasão Tumoral/imunologia , Animais , Antineoplásicos Imunológicos/farmacologia , Autofagia/genética , Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD8-Positivos/transplante , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Imunoterapia Adotiva , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Prognóstico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/terapia , Evasão Tumoral/genética
12.
PLoS One ; 15(8): e0237559, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32780783

RESUMO

BACKGROUND: The world is going through the critical phase of COVID-19 pandemic, caused by human coronavirus, SARS-CoV-2. Worldwide concerted effort to identify viral genomic changes across different sub-types has identified several strong changes in the coding region. However, there have not been many studies focusing on the variations in the 5' and 3' untranslated regions and their consequences. Considering the possible importance of these regions in host mediated regulation of viral RNA genome, we wanted to explore the phenomenon. METHODS: To have an idea of the global changes in 5' and 3'-UTR sequences, we downloaded 8595 complete and high-coverage SARS-CoV-2 genome sequence information from human host in FASTA format from Global Initiative on Sharing All Influenza Data (GISAID) from 15 different geographical regions. Next, we aligned them using Clustal Omega software and investigated the UTR variants. We also looked at the putative host RNA binding protein (RBP) and microRNA binding sites in these regions by 'RBPmap' and 'RNA22 v2' respectively. Expression status of selected RBPs and microRNAs were checked in lungs tissue. RESULTS: We identified 28 unique variants in SARS-CoV-2 UTR region based on a minimum variant percentage cut-off of 0.5. Along with 241C>T change the important 5'-UTR change identified was 187A>G, while 29734G>C, 29742G>A/T and 29774C>T were the most familiar variants of 3'UTR among most of the continents. Furthermore, we found that despite the variations in the UTR regions, binding of host RBP to them remains mostly unaltered, which further influenced the functioning of specific miRNAs. CONCLUSION: Our results, shows for the first time in SARS-Cov-2 infection, a possible cross-talk between host RBPs-miRNAs and viral UTR variants, which ultimately could explain the mechanism of escaping host RNA decay machinery by the virus. The knowledge might be helpful in developing anti-viral compounds in future.


Assuntos
Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Betacoronavirus/genética , Infecções por Coronavirus/metabolismo , Genoma Viral/genética , Instabilidade Genômica/genética , Interações Hospedeiro-Patógeno/genética , MicroRNAs/metabolismo , Pneumonia Viral/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Bases , Sítios de Ligação , Infecções por Coronavirus/virologia , Humanos , Fases de Leitura Aberta/genética , Pandemias , Pneumonia Viral/virologia , Ligação Proteica/genética
13.
Nat Commun ; 11(1): 3812, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732889

RESUMO

Vascular endothelial cell (EC) dysfunction plays a key role in diabetic complications. This study discovers significant upregulation of Quaking-7 (QKI-7) in iPS cell-derived ECs when exposed to hyperglycemia, and in human iPS-ECs from diabetic patients. QKI-7 is also highly expressed in human coronary arterial ECs from diabetic donors, and on blood vessels from diabetic critical limb ischemia patients undergoing a lower-limb amputation. QKI-7 expression is tightly controlled by RNA splicing factors CUG-BP and hnRNPM through direct binding. QKI-7 upregulation is correlated with disrupted cell barrier, compromised angiogenesis and enhanced monocyte adhesion. RNA immunoprecipitation (RIP) and mRNA-decay assays reveal that QKI-7 binds and promotes mRNA degradation of downstream targets CD144, Neuroligin 1 (NLGN1), and TNF-α-stimulated gene/protein 6 (TSG-6). When hindlimb ischemia is induced in diabetic mice and QKI-7 is knocked-down in vivo in ECs, reperfusion and blood flow recovery are markedly promoted. Manipulation of QKI-7 represents a promising strategy for the treatment of diabetic vascular complications.


Assuntos
Diabetes Mellitus Experimental/patologia , Células Endoteliais/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Doenças Vasculares/patologia , Animais , Antígenos CD/genética , Aterosclerose/patologia , Caderinas/genética , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular Neuronais/genética , Células Cultivadas , Regulação da Expressão Gênica/genética , Humanos , Hiperglicemia/patologia , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética
14.
Gene ; 761: 145037, 2020 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-32777526

RESUMO

Primordial germ cells (PGCs) are singled out from somatic cells very early during embryogenesis, then they migrate towards the genital ridge and differentiate into gametes through oogenesis or spermatogenesis. Labeling PGCs with Localized RNAexpression (LRE) technique by fluorescent proteins has been widely applied among teleost species to study the germ cell development and gonad differentiation. In this study, we first cloned and characterized the 3' untranslated regions (3'UTRs) of nanos homolog 1-like (nos1l), dead end (dnd), and vasa in yellow catfish (Pelteobagrus fulvidraco), and then synthesized the GFP-nos1l/dnd/vasa 3'UTR mRNAs. Each of these three 3'UTRs could label PGCs in yellow catfish embryos, of which, vasa 3'UTR exhibited the highest labeling efficiency. To identify the differences in PGCs at embryonic stage, XX all-female and XY all-male yellow catfish embryos were produced and injected with GFP-vasa 3'UTR mRNA. We observed the PGC migration route in these two monosex embryos from 24 hpf to 7 dpf, and found there was no difference between them. Besides, the PGC number was counted at 48 hpf, and the result showed that the average PGC number in XX females (11.3) was significantly larger than that in XY males (8.1).These findings provide an insight into the development of PGCs in yellow catfish embryos and the relationship between embryonicPGCnumberand thelatergonaddifferentiation.


Assuntos
Peixes-Gato/genética , Gametogênese/genética , Células Germinativas/metabolismo , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Movimento Celular/genética , RNA Helicases DEAD-box/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Gônadas/metabolismo , Masculino , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo
15.
Ecotoxicol Environ Saf ; 204: 111056, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32763566

RESUMO

Strontium (Sr) is an emerging environmental pollutant that has become a major global concern after the nuclear accident at the Fukushima Daiichi Nuclear Power Plant in 2011. Although many studies have demonstrated the harmful effects of Sr on plant growth and development at the physiological level, knowledge regarding how plants sense and respond to Sr stress at the molecular level is limited. Recent studies have suggested that microRNAs (miRNAs) function as key regulators of plant growth and development as well as in the responses of plants to environmental stresses, including salinity, drought, cold, nutrient starvation, and heavy metals. In this study, we examined the global expression profile of miRNAs under Sr stress using small RNA sequencing analysis in Arabidopsis to better understand the molecular basis of plant responses to Sr stress. To identify specific Sr-responsive miRNAs, we performed comparative miRNA expression profiling analysis using control, CaCl2-, and SrCl2-treated seedlings. Compared to the control treatment, the expressions of most miRNAs were considerably decreased in the Sr-treated seedlings. However, under Sr stress, the expressions of primary miRNAs (pri-miRNAs) and their target genes were significantly increased; the protein levels of HYPONASTIC LEAVES 1 (HYL1), one of the core components of the microprocessor complex, were strongly reduced despite the increased HYL1 mRNA expression. In addition, hyl1-2 mutant plants were shown to be more sensitive to Sr stress than wild-type plants. Collectively, our results strongly suggested that Sr stress may be associated with the disruption of miRNA biogenesis by reducing the protein level of HYL1, which is required to maintain proper growth and development for plants. Our findings further indicated that some miRNAs may play important roles in plant responses to Sr stress.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , MicroRNAs/biossíntese , Estresse Oxidativo/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Poluentes do Solo/toxicidade , Estrôncio/toxicidade , Arabidopsis/genética , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Estresse Oxidativo/genética , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Processamento Pós-Transcricional do RNA
16.
Virology ; 548: 59-72, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838947

RESUMO

Methylation of the N6 position of adenosine (m6A) is a widespread RNA modification that is critical for various physiological and pathological processes. Although this modification was also found in the RNA of several viruses almost 40 years ago, its biological functions during viral infection have been elucidated recently. Here, we investigated the effects of viral and host RNA methylation during porcine epidemic diarrhea virus (PEDV) infection. The results demonstrated that the m6A modification was abundant in the PEDV genome and the host methyltransferases METTL3 and METTL14 and demethylase FTO were involved in the regulation of viral replication. The knockdown of the methyltransferases increased PEDV replication while silencing the demethylase decreased PEDV output. Moreover, the proteins of the YTHDF family regulated the PEDV replication by affecting the stability of m6A-modified viral RNA. In particular, PEDV infection could trigger an increasement of m6A in host RNA and decrease the expression of FTO. The m6A modification sites in mRNAs and target genes were also altered during PEDV infection. Additionally, part of the host responses to PEDV infection was controlled by m6A modification, which could be reversed by the expression of FTO. Taken together, our results identified the role of m6A modification in PEDV replication and interactions with the host.


Assuntos
Adenosina/análogos & derivados , Infecções por Coronavirus/veterinária , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Vírus da Diarreia Epidêmica Suína/fisiologia , Doenças dos Suínos/genética , Doenças dos Suínos/virologia , Replicação Viral , Adenosina/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Genoma Viral , Metilação , Vírus da Diarreia Epidêmica Suína/ultraestrutura , Ligação Proteica , RNA Viral , Proteínas de Ligação a RNA/metabolismo , Suínos , Células Vero
17.
Nature ; 585(7823): 124-128, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848247

RESUMO

Tight coupling of transcription and translation is considered a defining feature of bacterial gene expression1,2. The pioneering ribosome can both physically associate and kinetically coordinate with RNA polymerase (RNAP)3-11, forming a signal-integration hub for co-transcriptional regulation that includes translation-based attenuation12,13 and RNA quality control2. However, it remains unclear whether transcription-translation coupling-together with its broad functional consequences-is indeed a fundamental characteristic of bacteria other than Escherichia coli. Here we show that RNAPs outpace pioneering ribosomes in the Gram-positive model bacterium Bacillus subtilis, and that this 'runaway transcription' creates alternative rules for both global RNA surveillance and translational control of nascent RNA. In particular, uncoupled RNAPs in B. subtilis explain the diminished role of Rho-dependent transcription termination, as well as the prevalence of mRNA leaders that use riboswitches and RNA-binding proteins. More broadly, we identified widespread genomic signatures of runaway transcription in distinct phyla across the bacterial domain. Our results show that coupled RNAP-ribosome movement is not a general hallmark of bacteria. Instead, translation-coupled transcription and runaway transcription constitute two principal modes of gene expression that determine genome-specific regulatory mechanisms in prokaryotes.


Assuntos
Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica , Biossíntese de Proteínas , Transcrição Genética , Regiões 5' não Traduzidas/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Filogenia , RNA Bacteriano/biossíntese , RNA Bacteriano/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator Rho/metabolismo , Ribossomos/metabolismo , Riboswitch/genética
18.
Nat Commun ; 11(1): 4305, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855391

RESUMO

Oligomeric assemblies of tau and the RNA-binding proteins (RBPs) Musashi (MSI) are reported in Alzheimer's disease (AD). However, the role of MSI and tau interaction in their aggregation process and its effects are nor clearly known in neurodegenerative diseases. Here, we investigated the expression and cellular localization of MSI1 and MSI2 in the brains tissues of Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) as well as in the wild-type mice and tau knock-out and P301L tau mouse models. We observed that formation of pathologically relevant protein inclusions was driven by the aberrant interactions between MSI and tau in the nuclei associated with age-dependent extracellular depositions of tau/MSI complexes. Furthermore, tau and MSI interactions induced impairment of nuclear/cytoplasm transport, chromatin remodeling and nuclear lamina formation. Our findings provide mechanistic insight for pathological accumulation of MSI/tau aggregates providing a potential basis for therapeutic interventions in neurodegenerative proteinopathies.


Assuntos
Núcleo Celular/patologia , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/patologia , Proteínas de Ligação a RNA/metabolismo , Proteínas tau/metabolismo , Transporte Ativo do Núcleo Celular , Idoso , Idoso de 80 Anos ou mais , Animais , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina , Citoplasma/metabolismo , Modelos Animais de Doenças , Feminino , Lobo Frontal/citologia , Lobo Frontal/patologia , Células HEK293 , Humanos , Corpos de Inclusão/patologia , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Agregados Proteicos , Ligação Proteica , Proteínas tau/genética
19.
Nat Commun ; 11(1): 3542, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669547

RESUMO

Ribosome biogenesis is an elaborate and energetically expensive program that involve two hundred protein factors in eukaryotes. Nuclear export of pre-ribosomal particles is one central step which also serves as an internal structural checkpoint to ensure the proper completion of nuclear assembly events. Here we present four structures of human pre-60S particles isolated through a nuclear export factor NMD3, representing assembly stages immediately before and after nuclear export. These structures reveal locations of a dozen of human factors, including an uncharacterized factor TMA16 localized between the 5S RNA and the P0 stalk. Comparison of these structures shows a progressive maturation for the functional regions, such as peptidyl transferase centre and peptide exit tunnel, and illustrate a sequence of factor-assisted rRNA maturation events. These data facilitate our understanding of the global conservation of ribosome assembly in eukaryotes and species-specific features of human assembly factors.


Assuntos
Núcleo Celular/metabolismo , Modelos Moleculares , RNA Ribossômico 5S/ultraestrutura , Proteínas Ribossômicas/ultraestrutura , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Microscopia Crioeletrônica , Humanos , RNA Ribossômico 5S/isolamento & purificação , RNA Ribossômico 5S/metabolismo , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Proteínas Ribossômicas/isolamento & purificação , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/ultraestrutura
20.
Nat Commun ; 11(1): 3751, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719344

RESUMO

The protein composition and structure of assembling 60S ribosomal subunits undergo numerous changes as pre-ribosomes transition from the nucleolus to the nucleoplasm. This includes stable anchoring of the Rpf2 subcomplex containing 5S rRNA, rpL5, rpL11, Rpf2 and Rrs1, which initially docks onto the flexible domain V of rRNA at earlier stages of assembly. In this work, we tested the function of the C-terminal domain (CTD) of Rpf2 during these anchoring steps, by truncating this extension and assaying effects on middle stages of subunit maturation. The rpf2Δ255-344 mutation affects proper folding of rRNA helices H68-70 during anchoring of the Rpf2 subcomplex. In addition, several assembly factors (AFs) are absent from pre-ribosomes or in altered conformations. Consequently, major remodeling events fail to occur: rotation of the 5S RNP, maturation of the peptidyl transferase center (PTC) and the nascent polypeptide exit tunnel (NPET), and export of assembling subunits to the cytoplasm.


Assuntos
Ribonucleoproteínas/metabolismo , Subunidades Ribossômicas Maiores/metabolismo , Rotação , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação/genética , Domínios Proteicos , Dobramento de Proteína , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Subunidades Ribossômicas Maiores/química , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura
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