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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.
Int J Mol Med ; 47(4): 1, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33604677

RESUMO

The Fos proto­oncogene, activator protein­1 (AP­1) transcription factor subunit (c­fos) gene, a member of the immediate early gene family, encodes c­Fos, which is a subunit of the AP­1 transcription factor. The present study aimed to investigate the mechanism by which the translation efficiency of c­fos mRNA is upregulated when cellular protein synthesis is shut off. The result of western blotting revealed that the protein expression levels of c­Fos were increased in rhabdomyosarcoma cells infected with enterovirus 71 (EV71) compared with uninfected cells. PCR was used to get the c­fos 5'­untranslated region (UTR). The luciferase assay of a bicistronic vector containing the c­fos 5'UTR revealed that the c­fos 5'UTR contains an internal ribosome entry site (IRES) sequence and a 175 nucleotide sequence (between 31 and 205 nt) that is essential for IRES activity. Analysis of potential IRES trans­acting factors revealed that poly(C)­binding protein 2 (PCBP2) negatively regulated the activity of the c­fos IRES, whereas the La autoantigen (La) positively regulated its activity. The results of RNA­protein immunoprecipitation demonstrated that both PCBP2 and La bound to the c­fos 5'UTR. Furthermore, the IRES activity of in vitro­transcribed c­fos mRNA was upregulated during EV71 infection. The present study suggested a mechanism for the effect of viral infection on host genes, and provided a novel target for gene translation regulation.


Assuntos
Regiões 5' não Traduzidas/genética , Regulação da Expressão Gênica/genética , Genes fos/genética , Sítios Internos de Entrada Ribossomal/genética , Proteínas Proto-Oncogênicas c-fos/genética , Autoantígenos/metabolismo , Sequência de Bases/genética , Linhagem Celular Tumoral , Enterovirus Humano A/metabolismo , Células HEK293 , Células HeLa , Humanos , Biossíntese de Proteínas/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/virologia , Ribonucleoproteínas/metabolismo , Ribossomos/metabolismo , Fator de Transcrição AP-1/genética , Transcrição Genética/genética
3.
Nat Commun ; 12(1): 880, 2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33563981

RESUMO

L1 retrotransposons can pose a threat to genome integrity. The host has evolved to restrict L1 replication. However, mechanisms underlying L1 propagation out of the host surveillance remains unclear. Here, we propose an evolutionary survival strategy of L1, which exploits RNA m6A modification. We discover that m6A 'writer' METTL3 facilitates L1 retrotransposition, whereas m6A 'eraser' ALKBH5 suppresses it. The essential m6A cluster that is located on L1 5' UTR serves as a docking site for eukaryotic initiation factor 3 (eIF3), enhances translational efficiency and promotes the formation of L1 ribonucleoprotein. Furthermore, through the comparative analysis of human- and primate-specific L1 lineages, we find that the most functional m6A motif-containing L1s have been positively selected and became a distinctive feature of evolutionarily young L1s. Thus, our findings demonstrate that L1 retrotransposons hijack the RNA m6A modification system for their successful replication.


Assuntos
Adenosina/análogos & derivados , Evolução Molecular , Elementos Nucleotídeos Longos e Dispersos/genética , RNA/metabolismo , Regiões 5' não Traduzidas , Adenosina/genética , Adenosina/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Animais , Células HeLa , Humanos , Metilação , Metiltransferases/metabolismo , Primatas/classificação , Primatas/genética , Biossíntese de Proteínas , RNA/química , Ribonucleoproteínas/metabolismo
4.
RNA Biol ; 18(2): 259-274, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33522422

RESUMO

La-related proteins (LARPs) share a La motif (LaM) followed by an RNA recognition motif (RRM). Together these are termed the La-module that, in the prototypical nuclear La protein and LARP7, mediates binding to the UUU-3'OH termination motif of nascent RNA polymerase III transcripts. We briefly review La and LARP7 activities for RNA 3' end binding and protection from exonucleases before moving to the more recently uncovered poly(A)-related activities of LARP1 and LARP4. Two features shared by LARP1 and LARP4 are direct binding to poly(A) and to the cytoplasmic poly(A)-binding protein (PABP, also known as PABPC1). LARP1, LARP4 and other proteins involved in mRNA translation, deadenylation, and decay, contain PAM2 motifs with variable affinities for the MLLE domain of PABP. We discuss a model in which these PABP-interacting activities contribute to poly(A) pruning of active mRNPs. Evidence that the SARS-CoV-2 RNA virus targets PABP, LARP1, LARP 4 and LARP 4B to control mRNP activity is also briefly reviewed. Recent data suggests that LARP4 opposes deadenylation by stabilizing PABP on mRNA poly(A) tails. Other data suggest that LARP1 can protect mRNA from deadenylation. This is dependent on a PAM2 motif with unique characteristics present in its La-module. Thus, while nuclear La and LARP7 stabilize small RNAs with 3' oligo(U) from decay, LARP1 and LARP4 bind and protect mRNA 3' poly(A) tails from deadenylases through close contact with PABP.Abbreviations: 5'TOP: 5' terminal oligopyrimidine, LaM: La motif, LARP: La-related protein, LARP1: La-related protein 1, MLLE: mademoiselle, NTR: N-terminal region, PABP: cytoplasmic poly(A)-binding protein (PABPC1), Pol III: RNA polymerase III, PAM2: PABP-interacting motif 2, PB: processing body, RRM: RNA recognition motif, SG: stress granule.


Assuntos
Autoantígenos/metabolismo , Poli A , Proteínas de Ligação a Poli(A)/metabolismo , Ribonucleoproteínas/metabolismo , Motivos de Aminoácidos , Humanos , Filogenia , Ligação Proteica , Biossíntese de Proteínas , Domínios Proteicos , Estabilidade de RNA , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , /genética
5.
Nat Commun ; 12(1): 1298, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33637717

RESUMO

Uridylation is a widespread modification destabilizing eukaryotic mRNAs. Yet, molecular mechanisms underlying TUTase-mediated mRNA degradation remain mostly unresolved. Here, we report that the Arabidopsis TUTase URT1 participates in a molecular network connecting several translational repressors/decapping activators. URT1 directly interacts with DECAPPING 5 (DCP5), the Arabidopsis ortholog of human LSM14 and yeast Scd6, and this interaction connects URT1 to additional decay factors like DDX6/Dhh1-like RNA helicases. Nanopore direct RNA sequencing reveals a global role of URT1 in shaping poly(A) tail length, notably by preventing the accumulation of excessively deadenylated mRNAs. Based on in vitro and in planta data, we propose a model that explains how URT1 could reduce the accumulation of oligo(A)-tailed mRNAs both by favoring their degradation and because 3' terminal uridines intrinsically hinder deadenylation. Importantly, preventing the accumulation of excessively deadenylated mRNAs avoids the biogenesis of illegitimate siRNAs that silence endogenous mRNAs and perturb Arabidopsis growth and development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , RNA Nucleotidiltransferases/metabolismo , RNA Interferente Pequeno/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Correpressoras/metabolismo , RNA Helicases DEAD-box/metabolismo , Regulação da Expressão Gênica de Plantas , Humanos , Proteínas Proto-Oncogênicas/metabolismo , RNA Nucleotidiltransferases/genética , Estabilidade de RNA/genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Tabaco/genética , Transcriptoma , Uridina/metabolismo
6.
Cell Mol Life Sci ; 78(6): 2607-2619, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33388834

RESUMO

Transfer RNA (tRNA)-derived fragments (tRFs) are an emerging class of conserved small non-coding RNAs that play important roles in post-transcriptional gene regulation. High-throughput sequencing of multiple biological samples have identified heterogeneous species of tRFs with distinct functionalities. These small RNAs have garnered a lot of scientific attention due to their ubiquitous expression and versatility in regulating various biological processes. In this review, we highlight our current understanding of tRF biogenesis and their regulatory functions. We summarize the diverse modes of biogenesis through which tRFs are generated and discuss the mechanism through which different tRF species regulate gene expression and the biological implications. Finally, we conceptualize research areas that require focus to strengthen our understanding of the biogenesis and function of tRFs.


Assuntos
Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/metabolismo , Animais , Regulação da Expressão Gênica , Humanos , Neoplasias/genética , Neoplasias/patologia , Plantas/genética , Plantas/metabolismo , Processamento Pós-Transcricional do RNA , Pequeno RNA não Traduzido/genética , RNA de Transferência/genética , Ribonucleoproteínas/metabolismo
7.
Nucleic Acids Res ; 49(2): 1114-1132, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33398331

RESUMO

The mitochondrial transcription termination factor proteins are nuclear-encoded nucleic acid binders defined by degenerate tandem helical-repeats of ∼30 amino acids. They are found in metazoans and plants where they localize in organelles. In higher plants, the mTERF family comprises ∼30 members and several of these have been linked to plant development and response to abiotic stress. However, knowledge of the molecular basis underlying these physiological effects is scarce. We show that the Arabidopsis mTERF9 protein promotes the accumulation of the 16S and 23S rRNAs in chloroplasts, and interacts predominantly with the 16S rRNA in vivo and in vitro. Furthermore, mTERF9 is found in large complexes containing ribosomes and polysomes in chloroplasts. The comprehensive analysis of mTERF9 in vivo protein interactome identified many subunits of the 70S ribosome whose assembly is compromised in the null mterf9 mutant, putative ribosome biogenesis factors and CPN60 chaperonins. Protein interaction assays in yeast revealed that mTERF9 directly interact with these proteins. Our data demonstrate that mTERF9 integrates protein-protein and protein-RNA interactions to promote chloroplast ribosomal assembly and translation. Besides extending our knowledge of mTERF functional repertoire in plants, these findings provide an important insight into the chloroplast ribosome biogenesis.


Assuntos
Proteínas de Arabidopsis/fisiologia , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Biogênese de Organelas , Fatores de Terminação de Peptídeos/fisiologia , RNA de Plantas/metabolismo , Ribonucleoproteínas/metabolismo , Ribossomos/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Polirribossomos/metabolismo , Biossíntese de Proteínas , RNA Ribossômico 16S/metabolismo , RNA Ribossômico 23S/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
8.
Nucleic Acids Res ; 49(2): 969-985, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33398341

RESUMO

Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T50 (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.


Assuntos
Sistemas CRISPR-Cas , Reparo do DNA por Junção de Extremidades , Edição de Genes , Vetores Genéticos/genética , Parvovirinae/genética , Reparo de DNA por Recombinação , Ribonucleoproteínas/metabolismo , Adulto , Linhagem Celular Tumoral , Variações do Número de Cópias de DNA , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Células HEK293 , Humanos , Ácidos Hidroxâmicos/farmacologia , Mutação INDEL , Células-Tronco Pluripotentes Induzidas , Cinética , RNA Guia/genética , Reparo de DNA por Recombinação/efeitos dos fármacos , Linfócitos T , Transdução Genética
9.
Methods Mol Biol ; 2167: 147-169, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32712919

RESUMO

Kink-turns are important RNA structural modules that facilitate long-range tertiary interactions and form binding sites for members of the L7Ae family of proteins. Present in a wide variety of functional RNAs, kink-turns play key organizational roles in many RNA-based cellular processes, including translation, modification, and tRNA biogenesis. It is important to determine the contribution of kink-turns to the overall architecture of resident RNAs, as these modules dictate ribonucleoprotein (RNP) assembly and function. This chapter describes a site-directed, hydroxyl radical-mediated footprinting strategy that utilizes L7Ae-tethered chemical nucleases to experimentally validate computationally identified kink-turns in any RNA and under a wide variety of conditions. The work plan described here uses the catalytic RNase P RNA as an example to provide a blueprint for using this footprinting method to map RNA-protein interactions in other RNP complexes.


Assuntos
Proteínas Arqueais/química , Pegada de DNA/métodos , Ácido Edético/análogos & derivados , Radical Hidroxila/química , Dobramento de RNA/genética , RNA/química , Ribonuclease P/metabolismo , Sítios de Ligação , Ácido Edético/química , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Ligação Proteica , RNA Catalítico/genética , RNA Catalítico/metabolismo , Transcrição Reversa , Ribonuclease P/genética , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Análise de Sequência de DNA
10.
Methods Mol Biol ; 2192: 69-73, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33230766

RESUMO

The incorporation of nucleoside analogs is a useful tool to study the various functions of DNA and RNA. These analogs can be detected directly by fluorescence or by immunolabeling, allowing to visualize, track, or measure the nucleic acid molecules in which they have been incorporated. In this chapter, methodologies to measure human mitochondrial transcription are described. The nascent RNA that is transcribed from mitochondrial DNA (mtDNA) has been shown to assemble into large ribonucleoprotein complexes that form discrete foci. These structures were called mitochondrial RNA granules (MRGs) and can be observed in vitro by the incorporation of a 5-Bromouridine (BrU), which is subsequently visualized by fluorescent immunolabeling. Here, a combined protocol for the MRGs detection is detailed, consisting of BrU labeling and visualization of one of their bona fide protein components, Fas-activated serine-threonine kinase domain 2 (FASTKD2). Based on immunodetection, the half-life and kinetics of the MRGs under various experimental conditions can further be determined by chasing the BrU pulse with an excess of Uridine.


Assuntos
Bromouracila/análogos & derivados , Imuno-Histoquímica/métodos , Complexos Multiproteicos/metabolismo , RNA Mitocondrial/metabolismo , Ribonucleoproteínas/metabolismo , Uridina/análogos & derivados , Bromouracila/metabolismo , DNA Mitocondrial/metabolismo , Meia-Vida , Células HeLa , Humanos , Cinética , Complexos Multiproteicos/química , Proteínas Serina-Treonina Quinases/metabolismo , Ribonucleoproteínas/química , Transcrição Genética , Uridina/metabolismo
11.
Nucleic Acids Res ; 49(5): e28, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33332543

RESUMO

RNA-protein interaction is central to post-transcriptional gene regulation. Identification of RNA-binding proteins relies mainly on UV-induced crosslinking (UVX) followed by the enrichment of RNA-protein conjugates and LC-MS/MS analysis. However, UVX has limited applicability in tissues of multicellular organisms due to its low penetration depth. Here, we introduce formaldehyde crosslinking (FAX) as an alternative chemical crosslinking for RNA interactome capture (RIC). Mild FAX captures RNA-protein interaction with high specificity and efficiency in cell culture. Unlike UVX-RIC, FAX-RIC robustly detects proteins that bind to structured RNAs or uracil-poor RNAs (e.g. AGO1, STAU1, UPF1, NCBP2, EIF4E, YTHDF proteins and PABP), broadening the coverage. Applied to Xenopus laevis oocytes and embryos, FAX-RIC provided comprehensive and unbiased RNA interactome, revealing dynamic remodeling of RNA-protein complexes. Notably, translation machinery changes during oocyte-to-embryo transition, for instance, from canonical eIF4E to noncanonical eIF4E3. Furthermore, using Mus musculus liver, we demonstrate that FAX-RIC is applicable to mammalian tissue samples. Taken together, we report that FAX can extend the RNA interactome profiling into multicellular organisms.


Assuntos
Proteômica/métodos , Ribonucleoproteínas/análise , Animais , Reagentes para Ligações Cruzadas , Embrião não Mamífero/metabolismo , Formaldeído , Células HeLa , Humanos , Fígado/metabolismo , Masculino , Camundongos , Oócitos/metabolismo , Peptídeos , Ribonucleoproteínas/metabolismo , Raios Ultravioleta , Xenopus laevis
12.
Methods Mol Biol ; 2170: 45-51, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797450

RESUMO

Northern blotting is a classical technique that allows the detection of specific nucleic acids using radioactive or non-radioactive probes. Normally, nucleic acids are denatured and separated by agarose or polyacrylamide gel electrophoresis and transferred and fixed to a membrane prior to detection. Here, we describe a method to analyze specific RNA in native ribonucleoprotein complexes using blue native PAGE with subsequent northern blotting, crosslinking of RNA onto a suitable membrane, and detection using non-radioactive probes.


Assuntos
Northern Blotting/métodos , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA/química , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo
13.
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
14.
Nat Microbiol ; 6(3): 339-353, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33349665

RESUMO

Characterizing the interactions that SARS-CoV-2 viral RNAs make with host cell proteins during infection can improve our understanding of viral RNA functions and the host innate immune response. Using RNA antisense purification and mass spectrometry, we identified up to 104 human proteins that directly and specifically bind to SARS-CoV-2 RNAs in infected human cells. We integrated the SARS-CoV-2 RNA interactome with changes in proteome abundance induced by viral infection and linked interactome proteins to cellular pathways relevant to SARS-CoV-2 infections. We demonstrated by genetic perturbation that cellular nucleic acid-binding protein (CNBP) and La-related protein 1 (LARP1), two of the most strongly enriched viral RNA binders, restrict SARS-CoV-2 replication in infected cells and provide a global map of their direct RNA contact sites. Pharmacological inhibition of three other RNA interactome members, PPIA, ATP1A1, and the ARP2/3 complex, reduced viral replication in two human cell lines. The identification of host dependency factors and defence strategies as presented in this work will improve the design of targeted therapeutics against SARS-CoV-2.


Assuntos
/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , /metabolismo , Autoantígenos/metabolismo , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Mapas de Interação de Proteínas , Proteoma , RNA Viral/genética , Ribonucleoproteínas/metabolismo , Replicação Viral/fisiologia
15.
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
16.
Mol Cell ; 80(1): 127-139.e6, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33007253

RESUMO

Human spliceosomes contain numerous proteins absent in yeast, whose functions remain largely unknown. Here we report a 3D cryo-EM structure of the human spliceosomal C complex at 3.4 Å core resolution and 4.5-5.7 Å at its periphery, and aided by protein crosslinking we determine its molecular architecture. Our structure provides additional insights into the spliceosome's architecture between the catalytic steps of splicing, and how proteins aid formation of the spliceosome's catalytically active RNP (ribonucleoprotein) conformation. It reveals the spatial organization of the metazoan-specific proteins PPWD1, WDR70, FRG1, and CIR1 in human C complexes, indicating they stabilize functionally important protein domains and RNA structures rearranged/repositioned during the Bact to C transition. Structural comparisons with human Bact, C∗, and P complexes reveal an intricate cascade of RNP rearrangements during splicing catalysis, with intermediate RNP conformations not found in yeast, and additionally elucidate the structural basis for the sequential recruitment of metazoan-specific spliceosomal proteins.


Assuntos
Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Spliceossomos/metabolismo , Animais , Catálise , Células HeLa , Humanos , Íntrons/genética , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Estabilidade Proteica , RNA/química , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Fatores de Tempo
17.
Nat Commun ; 11(1): 5041, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028827

RESUMO

Following introduction of CRISPR-Cas9 components into a cell, genome editing occurs unabated until degradation of its component nucleic acids and proteins by cellular processes. This uncontrolled reaction can lead to unintended consequences including off-target editing and chromosomal translocations. To address this, we develop a method for light-induced degradation of sgRNA termed CRISPRoff. Here we show that light-induced inactivation of ribonucleoprotein attenuates genome editing within cells and allows for titratable levels of editing efficiency and spatial patterning via selective illumination.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Luz , Estabilidade de RNA/efeitos da radiação , RNA Guia/metabolismo , Sistemas CRISPR-Cas/efeitos da radiação , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Estudos de Viabilidade , Células HEK293 , Humanos , RNA Guia/efeitos da radiação , Ribonucleoproteínas/metabolismo , Translocação Genética
18.
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
19.
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
20.
Nat Commun ; 11(1): 4355, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859915

RESUMO

The genome of influenza A viruses (IAV) is encoded in eight distinct viral ribonucleoproteins (vRNPs) that consist of negative sense viral RNA (vRNA) covered by the IAV nucleoprotein. Previous studies strongly support a selective packaging model by which vRNP segments are bundling to an octameric complex, which is integrated into budding virions. However, the pathway(s) generating a complete genome bundle is not known. We here use a multiplexed FISH assay to monitor all eight vRNAs in parallel in human lung epithelial cells. Analysis of 3.9 × 105 spots of colocalizing vRNAs provides quantitative insights into segment composition of vRNP complexes and, thus, implications for bundling routes. The complexes rarely contain multiple copies of a specific segment. The data suggest a selective packaging mechanism with limited flexibility by which vRNPs assemble into a complete IAV genome. We surmise that this flexibility forms an essential basis for the development of reassortant viruses with pandemic potential.


Assuntos
Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , RNA Viral/genética , Montagem de Vírus/genética , Montagem de Vírus/fisiologia , Células A549 , Células Epiteliais/virologia , Evolução Molecular , Humanos , Hibridização In Situ , Vírus da Influenza A Subtipo H3N2 , Influenza Humana/virologia , Pulmão , Modelos Teóricos , Ribonucleoproteínas/metabolismo
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