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Non-small cell lung cancer (NSCLC) remains one of the most common malignant tumors worldwide. The aim of the present study was to investigate the possibility of microRNA-20a (miR-20a) as a biomarker and therapeutic target for the diagnosis and treatment of NSCLC. Bioinformatics prediction, together with functional validation, confirmed miR-20a bound to programmed death ligand-1 (PD-L1) 3'-untranslated region to upregulate PD-L1 expression. Both miR-20a and PD-L1 could promote the proliferation of NSCLC cells. The expression level of PD-L1 was controlled by PTEN; however, further upstream regulation of PD-L1 expression was largely unknown. The present study showed that miR-20a could not restore the inhibition of PD-L1 expression levels by PTEN. Knockdown of PTEN expression upregulated the expression level of PD-L1 and promoted the proliferation of NSCLC cells. PTEN negatively regulated the Wnt/ß-catenin signaling pathway by inhibiting ß-catenin and Cyclin D1. Interestingly, PTEN could reverse miR-20a-mediated proliferation of NSCLC cells and the inhibitory effect was similar to that of XAV-939. miR-20a promotes the proliferation of NSCLC cells by inhibiting the expression level of PTEN and upregulating the expression level of PD-L1. It is suggested that miR-20a could be used as a biomarker and therapeutic target for the treatment of NSCLC.
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PURPOSE: Carbapenem-resistant Klebsiella pneumoniae (CRKP) represents a serious problem worldwide. Herein, we describe the evolution of ceftazidime-avibactam (CZA) resistance by sequencing clinical isolates from a patient with CRKP infection undergoing CZA treatment. PATIENTS AND METHODS: In this study, six CRKP strains were isolated from sputum and blood samples of a patient with CRKP infection after intracerebral hemorrhage. Two strains were selected for whole-genome analysis. RESULTS: Drug susceptibility testing showed that the MIC of CZA for CRKP strains isolated after 6 days of CZA treatment was 64-fold higher than that for three CRKP strains isolated before CZA treatment (4 vs >256 µg/mL), whereas the MIC of imipenem and meropenem was 128-fold (>32 vs 0.25 µg/mL) and 16-fold (> 32 vs 2 µg/mL) lower relatively, respectively. Multilocus sequence typing showed that all six CRKP strains isolated from the patient were ST11 and pulsed-field gel electrophoresis confirmed that they were of the same clone. Two strains were selected for whole-genome analysis. The aspartic acid residue at position 179 in the Ω loop was replaced by a tyrosine residue in the resistant strain, and the plasmid carried a bla KPC-2 to bla KPC-33 mutation. The results of the modified carbapenem inactivation method and the carbapenemase inhibitor enhancement and colloidal gold enzyme immunochromatographic assays for bla KPC-33 were negative. CONCLUSION: This is the first report from Henan to show that treatment with CZA for 6 days can cause mutations and change the phenotype from CZA sensitive to resistant. Therefore, routine testing for drug susceptibility and carbapenemase phenotypes should be conducted during treatment with CZA, and genotype determination is essential.
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BACKGROUND: With the increasing number of cases of breast cancer every year, the exploration of novel biomarkers has drawn attention. miR-331 has been demonstrated to play a role in various cancers, but its role in breast cancer is still unknown. METHODS: In this study, we included 121 patients with breast cancer treated at Affiliated Hospital of Weifang Medical University. Breast cancer tissues and adjacent normal tissues were collected during the surgery. The expression of miR-331 in breast cancer tissues and cell lines was detected by qRT-PCR assay. Then, with the help of Kaplan-Meier survival and Cox regression analyses, the role of miR-331 in the prognosis of breast cancer was analyzed. Finally, the effect of miR-331 on cell proliferation, migration, and invasion was investigated with CCK-8 assay and transwell assay. RESULTS: miR-331 was significantly upregulated in breast cancer tissues compared with normal tissues. The overexpression of miR-331 was associated with lymph node metastasis, TNM stage, and poor prognosis. From the results of Cox regression analyses, it was found that miR-331 served as an independent indicator in the prognosis of breast cancer. In addition, miR-331 was also found to be upregulated in breast cancer cells, which promoted cell proliferation, migration, and invasion of breast cancer. CONCLUSION: As shown from our data, miR-331 may be a potential prognostic biomarker in breast cancer. Moreover, the development and progression of breast cancer may involve miR-331. These findings suggest a novel therapeutic target for the treatment of breast cancer.
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Neoplasias de la Mama/patología , Progresión de la Enfermedad , MicroARNs/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Estimación de Kaplan-Meier , Metástasis Linfática , Invasividad Neoplásica , Pronóstico , Modelos de Riesgos Proporcionales , Regulación hacia ArribaRESUMEN
The current clinical protocol to conduct a bacterial antibiotic susceptibility test (AST) requires at least 18 hours, and cannot be accomplished during a single visit for patients. Here, a new method based on the technique of CRISPR-Cas12a is utilized to accomplish a bacterial genotypic AST within one hour with good accuracy. Two amplification approaches are employed and compared: (1) enriching the bacterial concentration by culturing in growth media; and (2) amplifying target DNA from raw samples by recombinase polymerase amplification (RPA). The results show that CRISPR combined with RPA can rapidly and accurately provide a bacterial genotypic AST of urine samples with urinary tract infections for precise antibiotic treatment. As such, this technology could open a new class of rapid bacterial genotypic AST for various infectious diseases.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Infecciones Urinarias , Antibacterianos/farmacología , Bacterias/genética , Sistemas CRISPR-Cas/genética , Humanos , Infecciones Urinarias/diagnóstico , Infecciones Urinarias/tratamiento farmacológicoRESUMEN
CRISPR-Cas immune systems utilize RNA-guided nucleases to protect bacteria from bacteriophage infection. Bacteriophages have in turn evolved inhibitory "anti-CRISPR" (Acr) proteins, including six inhibitors (AcrIIA1-AcrIIA6) that can block DNA cutting and genome editing by type II-A CRISPR-Cas9 enzymes. We show here that AcrIIA2 and its more potent homolog, AcrIIA2b, prevent Cas9 binding to DNA by occluding protein residues required for DNA binding. Cryo-EM-determined structures of AcrIIA2 or AcrIIA2b bound to S. pyogenes Cas9 reveal a mode of competitive inhibition of DNA binding that is distinct from other known Acrs. Differences in the temperature dependence of Cas9 inhibition by AcrIIA2 and AcrIIA2b arise from differences in both inhibitor structure and the local inhibitor-binding environment on Cas9. These findings expand the natural toolbox for regulating CRISPR-Cas9 genome editing temporally, spatially, and conditionally.
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Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , ADN/metabolismo , Edición Génica/métodos , Fagos Pseudomonas/metabolismo , Pseudomonas aeruginosa/enzimología , ARN Guía de Kinetoplastida/metabolismo , Temperatura , Proteínas Virales/metabolismo , Unión Competitiva , Proteína 9 Asociada a CRISPR/antagonistas & inhibidores , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/ultraestructura , Microscopía por Crioelectrón , ADN/genética , ADN/ultraestructura , Escherichia coli/enzimología , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Fagos Pseudomonas/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/virología , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/ultraestructura , Relación Estructura-Actividad , Proteínas Virales/genética , Proteínas Virales/ultraestructuraRESUMEN
Engineering of the Cpf1 crRNA has the potential to enhance its gene editing efficiency and non-viral delivery to cells. Here, we demonstrate that extending the length of its crRNA at the 5' end can enhance the gene editing efficiency of Cpf1 both in cells and in vivo. Extending the 5' end of the crRNA enhances the gene editing efficiency of the Cpf1 RNP to induce non-homologous end-joining and homology-directed repair using electroporation in cells. Additionally, chemical modifications on the extended 5' end of the crRNA result in enhanced serum stability. Also, extending the 5' end of the crRNA by 59 nucleotides increases the delivery efficiency of Cpf1 RNP in cells and in vivo cationic delivery vehicles including polymer nanoparticle. Thus, 5' extension and chemical modification of the Cpf1 crRNA is an effective method for enhancing the gene editing efficiency of Cpf1 and its delivery in vivo.
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Proteínas Bacterianas/metabolismo , Sistemas CRISPR-Cas/genética , Edición Génica , ARN Bacteriano/genética , Animales , Secuencia de Bases , Cationes , Reparación del ADN por Unión de Extremidades/genética , Células HEK293 , Células Hep G2 , Humanos , Lípidos/química , Ratones , Nanopartículas/química , Polímeros/química , Ribonucleoproteínas/metabolismoRESUMEN
The purpose of this study was to detect the expression of high-temperature requirement A2 (HtrA2) and its diagnostic value in the patients with hepatocellular carcinoma (HCC).The relative serum HtrA2 expression at mRNA and protein level was severally detected by quantitative real-time polymerase chain reaction and western blot analysis in 198 HCC patients and 48 healthy controls. And its association with clinicopathological features was analyzed by chi-square test. The diagnostic value of HtrA2 expression was estimated by establishing a receiver operating characteristic (ROC) curve.Serum HtrA2 was significantly higher in patients with HCC than that in healthy controls both at mRNA and protein levels (Pâ<â.05 for both). In addition, the high HtrA2 expression was associated with large tumor size and advanced clinical stage. Furthermore, the value of the area under the ROC curve was 0.808 corresponding with a sensitivity of 65.2% and a specificity of 89.6%, revealed that HtrA2 might be a diagnostic biomarker in HCC.HtrA2 is upregulated and considered to be a potential biomarker for the diagnosis of patients with HCC.
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Biomarcadores de Tumor/sangre , Carcinoma Hepatocelular/sangre , Serina Peptidasa A2 que Requiere Temperaturas Altas/sangre , Neoplasias Hepáticas/sangre , Adulto , Western Blotting , Carcinoma Hepatocelular/diagnóstico , Estudios de Casos y Controles , Femenino , Serina Peptidasa A2 que Requiere Temperaturas Altas/genética , Humanos , Neoplasias Hepáticas/diagnóstico , Masculino , ARN Mensajero/sangre , Curva ROC , Reacción en Cadena en Tiempo Real de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 gene editing technology is derived from a microbial adaptive immune system, where bacteriophages are often the intended target. Natural inhibitors of CRISPR-Cas9 enable phages to evade immunity and show promise in controlling Cas9-mediated gene editing in human cells. However, the mechanism of CRISPR-Cas9 inhibition is not known, and the potential applications for Cas9 inhibitor proteins in mammalian cells have not been fully established. We show that the anti-CRISPR protein AcrIIA4 binds only to assembled Cas9-single-guide RNA (sgRNA) complexes and not to Cas9 protein alone. A 3.9 Å resolution cryo-electron microscopy structure of the Cas9-sgRNA-AcrIIA4 complex revealed that the surface of AcrIIA4 is highly acidic and binds with a 1:1 stoichiometry to a region of Cas9 that normally engages the DNA protospacer adjacent motif. Consistent with this binding mode, order-of-addition experiments showed that AcrIIA4 interferes with DNA recognition but has no effect on preformed Cas9-sgRNA-DNA complexes. Timed delivery of AcrIIA4 into human cells as either protein or expression plasmid allows on-target Cas9-mediated gene editing while reducing off-target edits. These results provide a mechanistic understanding of AcrIIA4 function and demonstrate that inhibitors can modulate the extent and outcomes of Cas9-mediated gene editing.
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Proteína 9 Asociada a CRISPR/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/química , ADN/genética , Silenciador del Gen , Proteína 9 Asociada a CRISPR/química , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Línea Celular , Edición Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Modelos Moleculares , Conformación Molecular , ARN Guía de Kinetoplastida/genética , Relación Estructura-ActividadRESUMEN
Many bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems employ the dual RNA-guided DNA endonuclease Cas9 to defend against invading phages and conjugative plasmids by introducing site-specific double-stranded breaks in target DNA. Target recognition strictly requires the presence of a short protospacer adjacent motif (PAM) flanking the target site, and subsequent R-loop formation and strand scission are driven by complementary base pairing between the guide RNA and target DNA, Cas9-DNA interactions, and associated conformational changes. The use of CRISPR-Cas9 as an RNA-programmable DNA targeting and editing platform is simplified by a synthetic single-guide RNA (sgRNA) mimicking the natural dual trans-activating CRISPR RNA (tracrRNA)-CRISPR RNA (crRNA) structure. This review aims to provide an in-depth mechanistic and structural understanding of Cas9-mediated RNA-guided DNA targeting and cleavage. Molecular insights from biochemical and structural studies provide a framework for rational engineering aimed at altering catalytic function, guide RNA specificity, and PAM requirements and reducing off-target activity for the development of Cas9-based therapies against genetic diseases.
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Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , ADN/química , ARN/química , Proteínas Asociadas a CRISPR , ADN/genética , ARN/genética , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/genéticaRESUMEN
CRISPR/Cas9-based therapeutics, especially those that can correct gene mutations via homology directed repair (HDR), have the potential to revolutionize the treatment of genetic diseases. However, HDR-based therapeutics are challenging to develop because they require simultaneous in vivo delivery of Cas9 protein, guide RNA and donor DNA. Here, we demonstrate that a delivery vehicle composed of gold nanoparticles conjugated to DNA and complexed with cationic endosomal disruptive polymers can deliver Cas9 ribonucleoprotein and donor DNA into a wide variety of cell types, and efficiently correct the DNA mutation that causes Duchenne muscular dystrophy in mice via local injection, with minimal off-target DNA damage.
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Retinoic acid inducible gene-I (RIG-I) is a cytosolic pathogen recognition receptor that initiates the immune response against many RNA viruses. Upon RNA ligand binding, RIG-I undergoes a conformational change facilitating its homo-oligomerization and activation that results in its translocation from the cytosol to intracellular membranes to bind its signaling adaptor protein, mitochondrial antiviral-signaling protein (MAVS). Here we show that RIG-I activation is regulated by reversible acetylation. Acetyl-mimetic mutants of RIG-I do not form virus-induced homo-oligomers, revealing that acetyl-lysine residues of the RIG-I repressor domain prevent assembly to active homo-oligomers. During acute infection, deacetylation of RIG-I promotes its oligomerization upon ligand binding. We identify histone deacetylase 6 (HDAC6) as the deacetylase that promotes RIG-I activation and innate antiviral immunity to recognize and restrict RNA virus infection.
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Proteína 58 DEAD Box/metabolismo , Histona Desacetilasas/metabolismo , Acetilación/efectos de los fármacos , Animales , Bufexamac/farmacología , Línea Celular , Proteína 58 DEAD Box/antagonistas & inhibidores , Proteína 58 DEAD Box/genética , Genes Reporteros , Células HEK293 , Hepacivirus/genética , Hepacivirus/patogenicidad , Histona Desacetilasa 6 , Histona Desacetilasas/química , Histona Desacetilasas/genética , Humanos , Inmunidad Innata/efectos de los fármacos , Immunoblotting , Interferón beta/genética , Interferón beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo.
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Proteínas Bacterianas/metabolismo , Sistemas CRISPR-Cas , ADN/metabolismo , Endonucleasas/metabolismo , Edición Génica/métodos , Nucleosomas/metabolismo , Recombinación Genética , Animales , Proteína 9 Asociada a CRISPR , Unión Proteica , XenopusRESUMEN
Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding to form an RNA-DNA hybrid and a displaced DNA strand inside the protein. The role of this R-loop structure in positioning each DNA strand for cleavage by the two Cas9 nuclease domains is unknown. We determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions, in turn, position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30°, providing the structural distortion needed for R-loop formation.
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Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , División del ADN , ADN/química , Endonucleasas/química , Streptococcus pyogenes/enzimología , Dominio Catalítico , Cristalografía por Rayos X , Endonucleasas/ultraestructura , Ingeniería Genética , Genoma , Conformación de Ácido Nucleico , Conformación Proteica , ARN/química , ARN Guía de KinetoplastidaRESUMEN
RNAs with 5'-triphosphate (ppp) are detected in the cytoplasm principally by the innate immune receptor Retinoic Acid Inducible Gene-I (RIG-I), whose activation triggers a Type I IFN response. It is thought that self RNAs like mRNAs are not recognized by RIG-I because 5'ppp is capped by the addition of a 7-methyl guanosine (m7G) (Cap-0) and a 2'-O-methyl (2'-OMe) group to the 5'-end nucleotide ribose (Cap-1). Here we provide structural and mechanistic basis for exact roles of capping and 2'-O-methylation in evading RIG-I recognition. Surprisingly, Cap-0 and 5'ppp double-stranded (ds) RNAs bind to RIG-I with nearly identical Kd values and activate RIG-I's ATPase and cellular signaling response to similar extents. On the other hand, Cap-0 and 5'ppp single-stranded RNAs did not bind RIG-I and are signaling inactive. Three crystal structures of RIG-I complexes with dsRNAs bearing 5'OH, 5'ppp, and Cap-0 show that RIG-I can accommodate the m7G cap in a cavity created through conformational changes in the helicase-motif IVa without perturbing the ppp interactions. In contrast, Cap-1 modifications abrogate RIG-I signaling through a mechanism involving the H830 residue, which we show is crucial for discriminating between Cap-0 and Cap-1 RNAs. Furthermore, m7G capping works synergistically with 2'-O-methylation to weaken RNA affinity by 200-fold and lower ATPase activity. Interestingly, a single H830A mutation restores both high-affinity binding and signaling activity with 2'-O-methylated dsRNAs. Our work provides new structural insights into the mechanisms of host and viral immune evasion from RIG-I, explaining the complexity of cap structures over evolution.
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Guanosina/análogos & derivados , Inmunidad Innata , Caperuzas de ARN/metabolismo , ARN Helicasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencias de Aminoácidos , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Guanosina/química , Guanosina/metabolismo , Células HEK293 , Humanos , Hidrólisis , Metilación , Conformación de Ácido Nucleico , Estructura Terciaria de Proteína , ARN/química , ARN Bicatenario , Transducción de SeñalRESUMEN
RIG-I (Retinoic Acid Inducible Gene-I) is a cytosolic innate immune receptor that detects atypical features in viral RNAs as foreign to initiate a Type I interferon signaling response. RIG-I is present in an autoinhibited state in the cytoplasm and activated by blunt-ended double-stranded (ds)RNAs carrying a 5' triphosphate (ppp) moiety. These features found in many pathogenic RNAs are absent in cellular RNAs due to post-transcriptional modifications of RNA ends. Although RIG-I is structurally well characterized, the mechanistic basis for RIG-I's remarkable ability to discriminate between cellular and pathogenic RNAs is not completely understood. We show that RIG-I's selectivity for blunt-ended 5'-ppp dsRNAs is ≈3000 times higher than non-blunt ended dsRNAs commonly found in cellular RNAs. Discrimination occurs at multiple stages and signaling RNAs have high affinity and ATPase turnover rate and thus a high katpase/Kd. We show that RIG-I uses its autoinhibitory CARD2-Hel2i (second CARD-helicase insertion domain) interface as a barrier to select against non-blunt ended dsRNAs. Accordingly, deletion of CARDs or point mutations in the CARD2-Hel2i interface decreases the selectivity from ≈3000 to 150 and 750, respectively. We propose that the CARD2-Hel2i interface is a 'gate' that prevents cellular RNAs from generating productive complexes that can signal.
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ARN Helicasas DEAD-box/metabolismo , ARN/metabolismo , Adenosina Trifosfatasas/metabolismo , Secuencia de Bases , Sitios de Unión , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/genética , Polarización de Fluorescencia , Células HEK293 , Humanos , Interferón beta/genética , Interferón beta/metabolismo , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , ARN/química , ARN Bicatenario/metabolismo , Receptores InmunológicosRESUMEN
BACKGROUND: Detection of cervical high grade lesions in patients with atypical squamous cells of undetermined significance (ASCUS) is still a challenge. Our study tested the efficacy of the paired boxed gene 1 (PAX1) methylation analysis by methylation-sensitive high-resolution melting (MS-HRM) in the detection of high grade lesions in ASCUS and compared performance with the hybrid capture 2 (HC2) human papillomavirus (HPV) test. MATERIALS AND METHODS: A total of 463 consecutive ASCUS women from primary screening were selected. Their cervical scrapings were collected and assessed by PAX1 methylation analysis (MS-HRM) and high-risk HPV-DNA test (HC2). All patients with ASCUS were admitted to colposcopy and cervical biopsies. The Chi- square test was used to test the differences of PAX1 methylation or HPV infection between groups. RESULTS: The specificity, sensitivity, and accuracy for detecting CIN2 + lesions were: 95.6%, 82.4%, and 94.6%, respectively, for the PAX1 MS-HRM test; and 59.7%, 64.7%, and 60.0% for the HC2 HPV test. CONCLUSIONS: The PAX1 methylation analysis by MS-HRM demonstrated a better performance than the high-risk HPV-DNA test for the detection of high grade lesions (CIN2 +) in ASCUS cases. This approach could screen out the majority of low grade cases of ASCUS, and thus reduce the referral rate to colposcopy.
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Células Escamosas Atípicas del Cuello del Útero/patología , Metilación de ADN , Factores de Transcripción Paired Box/genética , Infecciones por Papillomavirus/genética , Displasia del Cuello del Útero/genética , Neoplasias del Cuello Uterino/genética , Adulto , Células Escamosas Atípicas del Cuello del Útero/metabolismo , Células Escamosas Atípicas del Cuello del Útero/virología , ADN Viral/genética , Femenino , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Estadificación de Neoplasias , Papillomaviridae/genética , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/patología , Infecciones por Papillomavirus/virología , Pronóstico , Neoplasias del Cuello Uterino/patología , Neoplasias del Cuello Uterino/virología , Displasia del Cuello del Útero/patología , Displasia del Cuello del Útero/virologíaRESUMEN
Bacterial adaptive immunity uses CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR transcripts for foreign DNA degradation. In type II CRISPR-Cas systems, activation of Cas9 endonuclease for DNA recognition upon guide RNA binding occurs by an unknown mechanism. Crystal structures of Cas9 bound to single-guide RNA reveal a conformation distinct from both the apo and DNA-bound states, in which the 10-nucleotide RNA "seed" sequence required for initial DNA interrogation is preordered in an A-form conformation. This segment of the guide RNA is essential for Cas9 to form a DNA recognition-competent structure that is poised to engage double-stranded DNA target sequences. We construe this as convergent evolution of a "seed" mechanism reminiscent of that used by Argonaute proteins during RNA interference in eukaryotes.
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Proteínas Argonautas/química , Proteínas Bacterianas/química , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , División del ADN , Endonucleasas/química , ARN Guía de Kinetoplastida/química , Streptococcus pyogenes/enzimología , Proteínas Bacterianas/genética , Secuencia de Bases , Proteína 9 Asociada a CRISPR , Cristalografía por Rayos X , ADN/química , Endonucleasas/genética , Activación Enzimática , Evolución Molecular , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Estructura Terciaria de Proteína , Interferencia de ARNRESUMEN
Prokaryotic CRISPR-Cas genomic loci encode RNA-mediated adaptive immune systems that bear some functional similarities with eukaryotic RNA interference. Acquired and heritable immunity against bacteriophage and plasmids begins with integration of â¼30 base pair foreign DNA sequences into the host genome. CRISPR-derived transcripts assemble with CRISPR-associated (Cas) proteins to target complementary nucleic acids for degradation. Here we review recent advances in the structural biology of these targeting complexes, with a focus on structural studies of the multisubunit Type I CRISPR RNA-guided surveillance and the Cas9 DNA endonuclease found in Type II CRISPR-Cas systems. These complexes have distinct structures that are each capable of site-specific double-stranded DNA binding and local helix unwinding.
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Proteínas Asociadas a CRISPR/química , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiología , Marcación de Gen/métodos , Modelos Moleculares , Microscopía por Crioelectrón , Cristalografía por Rayos X , Escherichia coliRESUMEN
Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.
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Actinomyces/enzimología , Proteínas Bacterianas/química , Endonucleasas/química , ARN/química , Streptococcus pyogenes/enzimología , Secuencia de Aminoácidos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Cristalografía por Rayos X , División del ADN , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Retinoic-acid-inducible gene-I (RIG-I; also known as DDX58) is a cytoplasmic pathogen recognition receptor that recognizes pathogen-associated molecular pattern (PAMP) motifs to differentiate between viral and cellular RNAs. RIG-I is activated by blunt-ended double-stranded (ds)RNA with or without a 5'-triphosphate (ppp), by single-stranded RNA marked by a 5'-ppp and by polyuridine sequences. Upon binding to such PAMP motifs, RIG-I initiates a signalling cascade that induces innate immune defences and inflammatory cytokines to establish an antiviral state. The RIG-I pathway is highly regulated and aberrant signalling leads to apoptosis, altered cell differentiation, inflammation, autoimmune diseases and cancer. The helicase and repressor domains (RD) of RIG-I recognize dsRNA and 5'-ppp RNA to activate the two amino-terminal caspase recruitment domains (CARDs) for signalling. Here, to understand the synergy between the helicase and the RD for RNA binding, and the contribution of ATP hydrolysis to RIG-I activation, we determined the structure of human RIG-I helicase-RD in complex with dsRNA and an ATP analogue. The helicase-RD organizes into a ring around dsRNA, capping one end, while contacting both strands using previously uncharacterized motifs to recognize dsRNA. Small-angle X-ray scattering, limited proteolysis and differential scanning fluorimetry indicate that RIG-I is in an extended and flexible conformation that compacts upon binding RNA. These results provide a detailed view of the role of helicase in dsRNA recognition, the synergy between the RD and the helicase for RNA binding and the organization of full-length RIG-I bound to dsRNA, and provide evidence of a conformational change upon RNA binding. The RIG-I helicase-RD structure is consistent with dsRNA translocation without unwinding and cooperative binding to RNA. The structure yields unprecedented insight into innate immunity and has a broader impact on other areas of biology, including RNA interference and DNA repair, which utilize homologous helicase domains within DICER and FANCM.