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1.
Int J Mol Sci ; 22(4)2021 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-33562320

RESUMO

Chloroplast ribonucleoproteins (cpRNPs) are nuclear-encoded and highly abundant proteins that are proposed to function in chloroplast RNA metabolism. However, the molecular mechanisms underlying the regulation of chloroplast RNAs involved in stress tolerance are poorly understood. Here, we demonstrate that CHLOROPLAST RNA-BINDING PROTEIN 1 (OsCRP1), a rice (Oryza sativa) cpRNP gene, is essential for stabilization of RNAs from the NAD(P)H dehydrogenase (NDH) complex, which in turn enhances drought and cold stress tolerance. An RNA-immunoprecipitation assay revealed that OsCRP1 is associated with a set of chloroplast RNAs. Transcript profiling indicated that the mRNA levels of genes from the NDH complex significantly increased in the OsCRP1 overexpressing compared to non-transgenic plants, whereas the pattern in OsCRP1 RNAi plants were opposite. Importantly, the OsCRP1 overexpressing plants showed a higher cyclic electron transport (CET) activity, which is essential for elevated levels of ATP for photosynthesis. Additionally, overexpression of OsCRP1 resulted in significantly enhanced drought and cold stress tolerance with higher ATP levels compared to wild type. Thus, our findings suggest that overexpression of OsCRP1 stabilizes a set of mRNAs from genes of the NDH complex involved in increasing CET activity and production of ATP, which consequently confers enhanced drought and cold tolerance.


Assuntos
Proteínas de Cloroplastos/metabolismo , Cloroplastos/genética , Temperatura Baixa , Secas , Oryza/crescimento & desenvolvimento , Estabilidade de RNA , Ribonucleoproteínas/metabolismo , Proteínas de Cloroplastos/genética , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Oryza/genética , Fotossíntese , Ribonucleoproteínas/genética , Estresse Fisiológico
2.
Methods Mol Biol ; 2209: 251-265, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33201474

RESUMO

In eukaryotic cells, aberrant mRNPs with processing and packaging defects are targeted co-transcriptionally by a surveillance system that triggers their nuclear retention and ultimately the degradation of their mRNA component by the 3'-5' activity of the exosome-associated exonuclease Rrp6. This mRNP quality control process is stimulated by the NNS complex (Nrd1-Nab3-Sen1), which otherwise mediates termination, processing, and decay of ncRNAs. The process involves also the exosome co-activator TRAMP complex (Trf4-Air2-Mtr4). Here, we describe a genome-wide approach to visualize the dynamic movement and coordination of these quality control components over the yeast chromosomes upon perturbation of mRNP biogenesis. The method provides valuable information on how the surveillance system is precisely coordinated both physically and functionally with the transcription machinery to detect the faulty events during perturbation of mRNP biogenesis. The overview shows also that the gathering of the quality control components over affected mRNA genes takes place at the expense of their commitment to be recruited at ncRNA genomic features, provoking termination and processing defects of ncRNAs.


Assuntos
RNA Fúngico/genética , RNA Mensageiro/genética , RNA não Traduzido/genética , Proteínas de Ligação a RNA/química , Ribonucleoproteínas/genética , Proteínas de Saccharomyces cerevisiae/química , Regulação Fúngica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Saccharomyces cerevisiae/genética , Transcrição Genética
3.
Methods Mol Biol ; 2162: 115-152, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32926381

RESUMO

CRISPR-Display uses the S. pyogenes Cas9 protein to posttranscriptionally localize noncoding RNA (ncRNA) domains to any genomic site, by directly fusing these domains to the Cas9 sgRNA cofactor. This versatile technology enables numerous applications for interrogating natural chromatin-regulatory ncRNAs, or for utilizing artificial ncRNA and ribonucleoprotein (RNP) devices at individual chromatin loci. To achieve these, a successful CRISPR-Display experiment requires that chimeric sgRNA-ncRNA fusions are stably expressed and incorporated into Cas9 complexes, and that they retain their ncRNA "cargo" domains at the targeted genomic sites. Here, I describe a workflow for designing, building, and testing such chimeric sgRNA-ncRNA expression constructs. I detail strategies for choosing expression systems and sgRNA insertion topologies, for assaying the incorporation of sgRNA-ncRNA fusions into functional Cas9 complexes, and for surveying the activities of ncRNA domains at targeted genomic loci. This establishes an initial set of "best practices" for the design and implementation of CRISPR-Display experiments.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Genômica/métodos , RNA Guia/genética , Proteína 9 Associada à CRISPR/genética , Regulação da Expressão Gênica/genética , Genoma/genética , Edição de RNA/genética , RNA não Traduzido/genética , Ribonucleoproteínas/genética
4.
Biochim Biophys Acta Mol Cell Res ; 1868(1): 118876, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33007331

RESUMO

Stress granules (SGs) are membrane-less ribonucleoprotein (RNP)-based cellular compartments that form in the cytoplasm of a cell upon exposure to various environmental stressors. SGs contain a large set of proteins, as well as mRNAs that have been stalled in translation as a result of stress-induced polysome disassembly. Despite the fact that SGs have been extensively studied for many years, their function is still not clear. They presumably help the cell to cope with the encountered stress, and facilitate the recovery process after stress removal upon which SGs disassemble. Aberrant formation of SGs and impaired SG disassembly majorly contribute to various pathological phenomena in cancer, viral infections, and neurodegeneration. The assembly of SGs is largely driven by liquid-liquid phase separation (LLPS), however, the molecular mechanisms behind that are not fully understood. Recent studies have proposed a novel mechanism for SG formation that involves the interplay of a large interaction network of mRNAs and proteins. Here, we review this novel concept of SG assembly, and discuss the current insights into SG disassembly.


Assuntos
Grânulos Citoplasmáticos/genética , Polirribossomos/genética , Ribonucleoproteínas/genética , Estresse Fisiológico/genética , Compartimento Celular/genética , Membrana Celular/genética , Citoplasma/genética , Humanos , Microextração em Fase Líquida , RNA Mensageiro/genética
5.
Methods Mol Biol ; 2211: 183-191, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33336278

RESUMO

The simple and versatile CRISPR/Cas9 system is a promising strategy for genome editing in mammalian cells. Generally, the genome editing components, namely Cas9 protein and single-guide RNA (sgRNA), are delivered in the format of plasmids, mRNA, or ribonucleoprotein (RNP) complexes. In particular, non-viral approaches are desirable as they overcome the safety concerns posed by viral vectors. To control cell fate for tissue regeneration, scaffold-based delivery of genome editing components will offer a route for local delivery and provide possible synergistic effects with other factors such as topographical cues that are co-delivered by the same scaffold. In this chapter, we detail a simple method of surface modification to functionalize electrospun nanofibers with CRISPR/Cas9 RNP complexes. The mussel-inspired bio-adhesive coating will be used as it is a simple and effective method to immobilize biomolecules on the surface. Nanofibers will provide a biomimicking microenvironment and topographical cues to seeded cells. For evaluation, a model cell line with single copies of enhanced green fluorescent protein (U2OS.EGFP) will be used to validate the efficiency of gene disruption.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Ribonucleoproteínas/metabolismo , Tecidos Suporte , Adesivos/química , Animais , Proteína 9 Associada à CRISPR/química , Linhagem Celular , Expressão Gênica , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lipídeos/química , Substâncias Macromoleculares/química , Substâncias Macromoleculares/metabolismo , Nanofibras/química , RNA Guia/química , RNA Guia/genética , Ribonucleoproteínas/genética , Engenharia Tecidual , Tecidos Suporte/química
6.
Nucleic Acids Res ; 49(1): 458-478, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33332560

RESUMO

The mammalian target of rapamycin (mTOR) is a critical regulator of cell growth, integrating multiple signalling cues and pathways. Key among the downstream activities of mTOR is the control of the protein synthesis machinery. This is achieved, in part, via the co-ordinated regulation of mRNAs that contain a terminal oligopyrimidine tract (TOP) at their 5'ends, although the mechanisms by which this occurs downstream of mTOR signalling are still unclear. We used RNA-binding protein (RBP) capture to identify changes in the protein-RNA interaction landscape following mTOR inhibition. Upon mTOR inhibition, the binding of LARP1 to a number of mRNAs, including TOP-containing mRNAs, increased. Importantly, non-TOP-containing mRNAs bound by LARP1 are in a translationally-repressed state, even under control conditions. The mRNA interactome of the LARP1-associated protein PABPC1 was found to have a high degree of overlap with that of LARP1 and our data show that PABPC1 is required for the association of LARP1 with its specific mRNA targets. Finally, we demonstrate that mRNAs, including those encoding proteins critical for cell growth and survival, are translationally repressed when bound by both LARP1 and PABPC1.


Assuntos
Autoantígenos/fisiologia , Proteína I de Ligação a Poli(A)/fisiologia , Polirribossomos/metabolismo , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/metabolismo , Ribonucleoproteínas/fisiologia , Serina-Treonina Quinases TOR/fisiologia , Regiões 5' não Traduzidas/genética , Autoantígenos/genética , Regulação da Expressão Gênica , Genes Reporter , Células HeLa , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 2 de Rapamicina/antagonistas & inibidores , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Naftiridinas/farmacologia , Mutação Puntual , Biossíntese de Proteínas/genética , Interferência de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas/genética
7.
J Vis Exp ; (166)2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33369608

RESUMO

The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been harnessed by researchers to study important biologically relevant problems. The unparalleled power of the CRISPR/Cas genome editing method allows researchers to precisely edit any locus of their choosing, thereby facilitating an increased understanding of gene function. Several methods for editing the C. elegans genome by CRISPR/Cas9 have been described previously. Here, we discuss and demonstrate a method which utilizes in vitro assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. Specifically, in this article, we go through the step-by-step process of introducing premature stop codons into the C. elegans rbm-3.2 gene by homology-directed repair using this method of CRISPR/Cas9 editing. This relatively simple editing method can be used to study the function of any gene of interest and allows for the generation of homozygous-edited C. elegans by CRISPR/Cas9 editing in less than two weeks.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Colágeno/genética , Testes Genéticos , Ribonucleoproteínas/metabolismo , Animais , Sequência de Bases , Primers do DNA/metabolismo , Gônadas/metabolismo , Homozigoto , Microinjeções , Edição de RNA/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Mapeamento por Restrição , Ribonucleoproteínas/genética , Streptococcus pyogenes/genética
8.
Int J Mol Sci ; 21(24)2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-33339270

RESUMO

Congenital cataracts are the prime cause for irreversible blindness in children. The global incidence of congenital cataract is 2.2-13.6 per 10,000 births, with the highest prevalence in Asia. Nearly half of the congenital cataracts are of familial nature, with a predominant autosomal dominant pattern of inheritance. Over 38 of the 45 mapped loci for isolated congenital or infantile cataracts have been associated with a mutation in a specific gene. The clinical and genetic heterogeneity of congenital cataracts makes the molecular diagnosis a bit of a complicated task. Hence, whole exome sequencing (WES) was utilized to concurrently screen all known cataract genes and to examine novel candidate factors for a disease-causing mutation in probands from 11 pedigrees affected with familial congenital cataracts. Analysis of the WES data for known cataract genes identified causative mutations in six pedigrees (55%) in PAX6, FYCO1 (two variants), EPHA2, P3H2,TDRD7 and an additional likely causative mutation in a novel gene NCOA6, which represents the first dominant mutation in this gene. This study identifies a novel cataract gene not yet linked to human disease. NCOA6 is a transcriptional coactivator that interacts with nuclear hormone receptors to enhance their transcriptional activator function.


Assuntos
Alelos , Catarata/genética , Coativadores de Receptor Nuclear/genética , Catarata/patologia , Efrina-A2/genética , Feminino , Testes Genéticos , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/genética , Mutação , Fator de Transcrição PAX6/genética , Linhagem , Pró-Colágeno-Prolina Dioxigenase/genética , Ribonucleoproteínas/genética , Sequenciamento Completo do Genoma
9.
Nucleic Acids Res ; 48(18): 10428-10440, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32960265

RESUMO

Cellular exonucleases involved in the processes that regulate RNA stability and quality control have been shown to restrict or to promote the multiplication cycle of numerous RNA viruses. Influenza A viruses are major human pathogens that are responsible for seasonal epidemics, but the interplay between viral proteins and cellular exonucleases has never been specifically studied. Here, using a stringent interactomics screening strategy and an siRNA-silencing approach, we identified eight cellular factors among a set of 75 cellular proteins carrying exo(ribo)nuclease activities or involved in RNA decay processes that support influenza A virus multiplication. We show that the exoribonuclease ERI1 interacts with the PB2, PB1 and NP components of the viral ribonucleoproteins and is required for viral mRNA transcription. More specifically, we demonstrate that the protein-protein interaction is RNA dependent and that both the RNA binding and exonuclease activities of ERI1 are required to promote influenza A virus transcription. Finally, we provide evidence that during infection, the SLBP protein and histone mRNAs co-purify with vRNPs alongside ERI1, indicating that ERI1 is most probably recruited when it is present in the histone pre-mRNA processing complex in the nucleus.


Assuntos
Exorribonucleases/genética , Vírus da Influenza A/genética , Influenza Humana/genética , Proteínas Nucleares/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Linhagem Celular , Histonas/genética , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/patogenicidade , Influenza Humana/virologia , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Interferente Pequeno , RNA Viral/genética , Ribonucleoproteínas/genética , Transcrição Genética/genética , Proteínas Virais/genética , Replicação Viral/genética
10.
Sci Rep ; 10(1): 14733, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32895447

RESUMO

Nitazoxanide (NTZ) is effective against helminths and numerous microorganisms, including bacteria and viruses. In vivo, NTZ is metabolized into Tizoxanide (TIZ), which is the active circulating metabolite. With the emergence of SARS-Cov-2 as a Pandemic agent, NTZ became one of the molecules already approved for human use to engage clinical trials, due to results in vitro showing that NTZ was highly effective against the SARS-Cov-2, agent of COVID-19. There are currently several ongoing clinical trials mainly in the USA and Brazil involving NTZ due not only to the in vitro results, but also for its long-known safety. Here, we study the response of Vero cells to TIZ treatment and unveil possible mechanisms for its antimicrobial effect, using a label-free proteomic approach (LC/MS/MS) analysis to compare the proteomic profile between untreated- and TIZ-treated cells. Fifteen differentially expressed proteins were observed related to various biological processes, including translation, intracellular trafficking, RNA processing and modification, and signal transduction. The broad antimicrobial range of TIZ points towards its overall effect in lowering cell metabolism and RNA processing and modification. The decreased levels of FASN, HNRNPH and HNRNPK with the treatment appear to be important for antiviral activity.


Assuntos
Anti-Infecciosos/farmacologia , Proteoma/efeitos dos fármacos , Tiazóis/farmacologia , Animais , Chlorocebus aethiops , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Proteoma/genética , Proteoma/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Células Vero
11.
PLoS Pathog ; 16(8): e1008326, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32804988

RESUMO

CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with cotransformation of transiently stable vectors containing telomeres, which allowed temporary selection and convenient screening for marker-free editing events. We demonstrate that this approach provides superior editing rates compared to existing CRISPR/Cas-based methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae. Genome sequencing of edited strains revealed very few additional mutations and no evidence for RNP-mediated off-targeting. The high performance of telomere vector-mediated editing was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels towards different SDHI fungicides. The increased efficiency and easy handling of RNP-based cotransformation is expected to accelerate molecular research in B. cinerea and other fungi.


Assuntos
Botrytis/fisiologia , Sistemas CRISPR-Cas , Edição de Genes , Oryza/microbiologia , Doenças das Plantas/microbiologia , Ribonucleoproteínas/antagonistas & inibidores , Telômero/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Oryza/genética , Doenças das Plantas/genética , Ribonucleoproteínas/genética
12.
Mol Cell ; 79(6): 991-1007.e4, 2020 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-32780990

RESUMO

Stress granules (SGs) are condensates of mRNPs that form in response to stress. SGs arise by multivalent protein-protein, protein-RNA, and RNA-RNA interactions. However, the role of RNA-RNA interactions in SG assembly remains understudied. Here, we describe a yeast SG reconstitution system that faithfully recapitulates SG assembly in response to trigger RNAs. SGs assembled by stem-loop RNA triggers are ATP-sensitive, regulated by helicase/chaperone activity, and exhibit the hallmarks of maturation observed for SG proteins that phase-separate in vitro. Additionally, the fraction of total RNA that phase-separates in vitro is sufficient to trigger SG formation. However, condensation of NFT1 mRNA, an enriched transcript in this population, can only assemble an incomplete SG. These results suggest that networks of distinct transcripts are required to form a canonical SG and provide a platform for dissecting the interplay between the transcriptome and ATP-dependent remodeling in SG formation.


Assuntos
Grânulos Citoplasmáticos/genética , Ribonucleoproteínas/genética , Estresse Fisiológico/genética , Transcriptoma/genética , Trifosfato de Adenosina/genética , Linhagem Celular , Regulação Fúngica da Expressão Gênica/genética , Humanos , RNA/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética
13.
Biomolecules ; 10(8)2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32784769

RESUMO

The ribonome interconnects the proteome and the transcriptome. Specific biology is situated at this interface, which can be studied in bulk using omics approaches or specifically by targeting an individual protein or RNA species. In this review, we focus on both RNA- and ribonucleoprotein-(RNP) centric methods. These methods can be used to study the dynamics of the ribonome in response to a stimulus or to identify the proteins that interact with a specific RNA species. The purpose of this review is to provide and discuss an overview of strategies to cross-link RNA to proteins and the currently available RNA- and RNP-centric approaches to study RNPs. We elaborate on some major challenges common to most methods, involving RNP yield, purity and experimental cost. We identify the origin of these difficulties and propose to combine existing approaches to overcome these challenges. The solutions provided build on the recently developed organic phase separation protocols, such as Cross-Linked RNA eXtraction (XRNAX), orthogonal organic phase separation (OOPS) and Phenol-Toluol extraction (PTex).


Assuntos
Proteômica/métodos , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas de Ligação a RNA/metabolismo , RNA/isolamento & purificação , RNA/metabolismo , Ribonucleoproteínas/isolamento & purificação , Ribonucleoproteínas/metabolismo , Animais , Humanos , Ligação Proteica , Proteoma/metabolismo , RNA/genética , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Transcriptoma
14.
Nucleic Acids Res ; 48(16): 9320-9335, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32813011

RESUMO

Heterogeneous nuclear ribonuclear protein K (hnRNPK) is an abundant RNA-binding protein crucial for a wide variety of biological processes. While its binding preference for multi-cytosine-patch (C-patch) containing RNA is well documented, examination of binding to known cellular targets that contain C-patches reveals an unexpected breadth of binding affinities. Analysis of in-cell crosslinking data reinforces the notion that simple C-patch preference is not fully predictive of hnRNPK localization within transcripts. The individual RNA-binding domains of hnRNPK work together to interact with RNA tightly, with the KH3 domain being neither necessary nor sufficient for binding. Rather, the RG/RGG domain is implicated in providing essential contributions to RNA-binding, but not DNA-binding, affinity. hnRNPK is essential for X chromosome inactivation, where it interacts with Xist RNA specifically through the Xist B-repeat region. We use this interaction with an RNA motif derived from this B-repeat region to determine the RNA-structure dependence of C-patch recognition. While the location preferences of hnRNPK for C-patches are conformationally restricted within the hairpin, these structural constraints are relieved in the absence of RNA secondary structure. Together, these results illustrate how this multi-domain protein's ability to accommodate and yet discriminate between diverse cellular RNAs allows for its broad cellular functions.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Motivos de Nucleotídeos/genética , RNA Longo não Codificante/genética , Ribonucleoproteínas/genética , Animais , Pareamento de Bases/genética , Núcleo Celular/genética , Feminino , Humanos , Proteínas de Ligação a RNA/genética , Inativação do Cromossomo X/genética
15.
PLoS One ; 15(8): e0238254, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841293

RESUMO

The identification of host / pathogen interactions is essential to both understanding the molecular biology of infection and developing rational intervention strategies to overcome disease. Alphaviruses, such as Sindbis virus, Chikungunya virus, and Venezuelan Equine Encephalitis virus are medically relevant positive-sense RNA viruses. As such, they must interface with the host machinery to complete their infectious lifecycles. Nonetheless, exhaustive RNA:Protein interaction discovery approaches have not been reported for any alphavirus species. Thus, the breadth and evolutionary conservation of host interactions on alphaviral RNA function remains a critical gap in the field. Herein we describe the application of the Cross-Link Assisted mRNP Purification (CLAMP) strategy to identify conserved alphaviral interactions. Through comparative analyses, conserved alphaviral host / pathogen interactions were identified. Approximately 100 unique host proteins were identified as a result of these analyses. Ontological assessments reveal enriched Molecular Functions and Biological Processes relevant to alphaviral infection. Specifically, as anticipated, Poly(A) RNA Binding proteins are significantly enriched in virus specific CLAMP data sets. Moreover, host proteins involved in the regulation of mRNA stability, proteasome mediated degradation, and a number of 14-3-3 proteins were identified. Importantly, these data expand the understanding of alphaviral host / pathogen interactions by identifying conserved interactants.


Assuntos
Alphavirus/genética , Alphavirus/patogenicidade , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Proteínas de Ligação a Poli(A)/genética , Proteínas de Ligação a Poli(A)/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Alphavirus/fisiologia , Animais , Linhagem Celular , Vírus Chikungunya/genética , Vírus Chikungunya/patogenicidade , Vírus Chikungunya/fisiologia , Vírus da Encefalite Equina Venezuelana/genética , Vírus da Encefalite Equina Venezuelana/patogenicidade , Vírus da Encefalite Equina Venezuelana/fisiologia , Evolução Molecular , Células HEK293 , Humanos , Mapas de Interação de Proteínas , Ribonucleoproteínas/genética , Ribonucleoproteínas/isolamento & purificação , Ribonucleoproteínas/metabolismo , Vírus Sindbis/genética , Vírus Sindbis/patogenicidade , Vírus Sindbis/fisiologia , Especificidade da Espécie
16.
Proc Natl Acad Sci U S A ; 117(28): 16557-16566, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601201

RESUMO

Influenza viruses (IV) exploit a variety of signaling pathways. Previous studies showed that the rapidly accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway is functionally linked to nuclear export of viral ribonucleoprotein (vRNP) complexes, suggesting that vRNP export is a signaling-induced event. However, the underlying mechanism remained completely enigmatic. Here we have dissected the unknown molecular steps of signaling-driven vRNP export. We identified kinases RSK1/2 as downstream targets of virus-activated ERK signaling. While RSK2 displays an antiviral role, we demonstrate a virus-supportive function of RSK1, migrating to the nucleus to phosphorylate nucleoprotein (NP), the major constituent of vRNPs. This drives association with viral matrix protein 1 (M1) at the chromatin, important for vRNP export. Inhibition or knockdown of MEK, ERK or RSK1 caused impaired vRNP export and reduced progeny virus titers. This work not only expedites the development of anti-influenza strategies, but in addition demonstrates converse actions of different RSK isoforms.


Assuntos
Vírus da Influenza A/metabolismo , Influenza Humana/virologia , Ribonucleoproteínas/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Humanos , Vírus da Influenza A/genética , Influenza Humana/genética , Influenza Humana/metabolismo , Sistema de Sinalização das MAP Quinases , Sinais de Exportação Nuclear , Ribonucleoproteínas/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
17.
Nucleic Acids Res ; 48(15): 8626-8644, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32621609

RESUMO

The exon junction complex (EJC) is an essential constituent and regulator of spliced messenger ribonucleoprotein particles (mRNPs) in metazoans. As a core component of the EJC, CASC3 was described to be pivotal for EJC-dependent nuclear and cytoplasmic processes. However, recent evidence suggests that CASC3 functions differently from other EJC core proteins. Here, we have established human CASC3 knockout cell lines to elucidate the cellular role of CASC3. In the knockout cells, overall EJC composition and EJC-dependent splicing are unchanged. A transcriptome-wide analysis reveals that hundreds of mRNA isoforms targeted by nonsense-mediated decay (NMD) are upregulated. Mechanistically, recruiting CASC3 to reporter mRNAs by direct tethering or via binding to the EJC stimulates mRNA decay and endonucleolytic cleavage at the termination codon. Building on existing EJC-NMD models, we propose that CASC3 equips the EJC with the persisting ability to communicate with the NMD machinery in the cytoplasm. Collectively, our results characterize CASC3 as a peripheral EJC protein that tailors the transcriptome by promoting the degradation of EJC-dependent NMD substrates.


Assuntos
Proteínas de Neoplasias/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Processamento de RNA/genética , Proteínas de Ligação a RNA/genética , Transcriptoma/genética , Sequência de Aminoácidos/genética , Núcleo Celular/genética , Éxons/genética , Técnicas de Inativação de Genes , Humanos , RNA Mensageiro/genética , Ribonucleoproteínas/genética
18.
Yi Chuan ; 42(6): 556-564, 2020 Jun 20.
Artigo em Chinês | MEDLINE | ID: mdl-32694114

RESUMO

The CRISPR/Cas system is the most popular genome editing technology in recent years and has been widely used in crop improvement. Compared with introducing the CRISPR/Cas system into plant cells with DNA constructs, introducing CRISPR/Cas ribonucleoprotein (RNP) to perform genome editing excels in rapid action, low off-target rates and is free of DNA insertions in editing plants. However, efficient delivery of CRISPR/Cas RNP into plant cells and achieving high editing frequency are still very challenging, which limits the extensive implementation of CRISPR/Cas RNP-mediated genome editing in plants. In this review, we summarize the progress of protein and RNP delivery methods in plant cells, and provide new perspectives of further development and future applications of the CRISPR/Cas RNP technology in plant genome editing.


Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Sistemas CRISPR-Cas/genética , Genoma de Planta/genética , Plantas , Ribonucleoproteínas/genética
19.
Nucleic Acids Res ; 48(12): 6811-6823, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32496535

RESUMO

A key aim in exploiting CRISPR-Cas is gRNA engineering to introduce additional functionalities, ranging from individual nucleotide changes that increase efficiency of on-target binding to the inclusion of larger functional RNA aptamers or ribonucleoproteins (RNPs). Cas9-gRNA interactions are crucial for complex assembly, but several distinct regions of the gRNA are amenable to modification. We used in vitro ensemble and single-molecule assays to assess the impact of gRNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that RNP formation was unaffected by any of our modifications. R-loop formation and DNA cleavage activity were also essentially unaffected by modification of the Upper Stem, first Hairpin and 3' end. In contrast, we found that 5' additions of only two or three nucleotides could reduce R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such modifications are a common by-product of in vitro transcribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5' end of a gRNA still supported RNP formation but produced a stable ∼9 bp R-loop that could not activate DNA cleavage. Consideration of these observations will assist in successful gRNA design.


Assuntos
Sistemas CRISPR-Cas/genética , Clivagem do DNA , Estruturas R-Loop/genética , RNA Guia/genética , Aptâmeros de Nucleotídeos/genética , Edição de Genes , Conformação de Ácido Nucleico , RNA Guia/ultraestrutura , Ribonucleoproteínas/genética , Ribonucleoproteínas/ultraestrutura , Imagem Individual de Molécula , Streptococcus pyogenes/genética
20.
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
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