RESUMO
AIMS: Pathogenic bacteria can spread between individuals or between food items via the surfaces they share. Limiting the survival of pathogens on surfaces, therefore, presents an opportunity to limit at least one route of how pathogens spread. In this study, we propose that a simple coating with the essential oil isoeugenol can be used to circumvent the problem of bacterial transfer via surfaces. METHODS AND RESULTS: Two commonly used materials, stainless steel and polyethylene, were coated by physical adsorption, and the coatings were characterized by Raman spectroscopy, atomic force microscopy and water contact angle measurements. We quantified and visualized the colonization of coated and uncoated surfaces by three bacteria: Staphylococcus aureus, Listeria monocytogenes and Pseudomonas fluorescens. No viable cells were detected on surfaces coated with isoeugenol. CONCLUSIONS: The isoeugenol coating prepared with simple adsorption proved effective in preventing biofilm formation on stainless steel and polyethylene surfaces. The result was caused by the antibacterial effect of isoeugenol, as the coating did not diminish the adhesive properties of the surface. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study demonstrates that a simple isoeugenol coating can prevent biofilm formation of S. aureus, L. monocytogenes and P. fluorescens on two commonly used surfaces.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Eugenol/análogos & derivados , Listeria monocytogenes/fisiologia , Polietileno/química , Pseudomonas fluorescens/fisiologia , Staphylococcus aureus/fisiologia , Adsorção , Antibacterianos/química , Aderência Bacteriana/efeitos dos fármacos , Eugenol/química , Eugenol/farmacologia , Humanos , Listeria monocytogenes/efeitos dos fármacos , Pseudomonas fluorescens/efeitos dos fármacos , Aço Inoxidável/química , Staphylococcus aureus/efeitos dos fármacosRESUMO
Circular RNA (circRNA) is a novel member of the noncoding cancer genome with distinct properties and diverse cellular functions, which is being explored at a steadily increasing pace. The list of endogenous circRNAs involved in cancer continues to grow; however, the functional relevance of the vast majority is yet to be discovered. In general, circRNAs are exceptionally stable molecules and some have been shown to function as efficient microRNA sponges with gene-regulatory potential. Many circRNAs are highly conserved and have tissue-specific expression patterns, which often do not correlate well with host gene expression. Here we review the current knowledge on circRNAs in relation to their implications in tumorigenesis as well as their potential as diagnostic and prognostic biomarkers and as possible therapeutic targets in future personalized medicine. Finally, we discuss future directions for circRNA cancer research and current caveats, which must be addressed to facilitate the translation of basic circRNA research into clinical use.
Assuntos
Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias/genética , RNA/genética , Biomarcadores Tumorais/genética , Pesquisa Biomédica/métodos , Pesquisa Biomédica/tendências , Humanos , Oncologia/métodos , Oncologia/tendências , Neoplasias/sangue , Neoplasias/mortalidade , Neoplasias/terapia , Medicina de Precisão/métodos , Prognóstico , RNA CircularRESUMO
This work demonstrates gastric mucin-triggered nanocarrier disassembly for release of antisense oligonucleotides and consequent unassisted cellular entry as a novel oral delivery strategy. A fluorescence activation-based reporter system was used to investigate the interaction and mucin-mediated disassembly of chitosan-based nanocarriers containing a 13-mer DNA oligonucleotide with a flanked locked RNA nucleic acid gapmer design. Gastric mucins were shown to trigger gapmer release from nanocarriers that was dependent on the interaction time, mucin concentration and N : P ratio with a maximal release at N : P 10. In contrast to siRNA, naked gapmers exhibited uptake into mucus producing HT-MTX mono-cultures and HT-MTX co-cultured with the carcinoma epithelial cell line Caco-2. Importantly, in vivo gapmer uptake was observed in epithelial tissue 30 min post-injection in murine intestinal loops. The findings present a mucosal design-based system tailored for local delivery of oligonucleotides that may maximize the effectiveness of gene silencing therapeutics within tumours at mucosal sites.
Assuntos
Portadores de Fármacos , Mucinas/química , Nanopartículas , Oligonucleotídeos Antissenso/administração & dosagem , Animais , Células CACO-2 , Quitosana , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno , SuínosRESUMO
DNA origami has been used to orchestrate reactions with nano-precision using a variety of biomolecules. Here, the dynamics of albumin-assisted, localized single-molecule DNA polymerization by terminal deoxynucleotidyl transferase on a 2D DNA origami are monitored using AFM in liquid. Direct visualization of the surface activity revealed the mechanics of growth.
Assuntos
Biotecnologia/métodos , DNA Nucleotidilexotransferase/metabolismo , DNA/metabolismo , DNA/ultraestrutura , Nanoestruturas/ultraestrutura , Nanotecnologia/métodos , Albuminas , DNA/química , Microscopia de Força Atômica , Nanoestruturas/química , Polimerização , Propriedades de SuperfícieRESUMO
BACKGROUND: The tyrosine kinase receptor HER4 is a member of the epidermal growth factor receptor (EGFR) family. It plays diverse roles in cancer development and cancer progression and can both exert oncogenic and tumour-suppressive activities. Alternatively spliced isoforms of HER4 are critical to the different signalling possibilities of HER4. METHODS: We use a splice-switching oligonucleotide (SSO) to direct the alternative splicing of HER4 from the CYT1 to the CYT2 isoform in HER4-expressing breast cancer cells. RESULTS: Treatment with a target-specific SSO was accompanied by a decreased growth of the cells (P<0.0001). In addition, the SSO treatment induced a decreased activity of Akt. We confirmed the SSO-dependent switching of the HER4 isoform CYT1 to CYT2 expression in a xenografted mouse tumour model driven by subcutaneously injected MCF7 cells. We hence demonstrated the feasibility of SSO-directed splice-switching activity in vivo. Furthermore, the SSO treatment efficiently decreased the growth of the xenografted tumour (P=0.0014). CONCLUSION: An SSO directing the splicing of HER4 towards the CYT2 isoform has an inhibitory effect of cancer cell growth in vitro and in vivo. These results may pave the way for the development of new anticancer drugs in HER4-deregulated cancers in humans.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/terapia , Receptores ErbB/genética , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/biossíntese , Processamento Alternativo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Receptores ErbB/biossíntese , Feminino , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Células MCF-7 , Camundongos , Camundongos Endogâmicos C3H , Camundongos Nus , Oligonucleotídeos Antissenso/genética , RNA Mensageiro/genética , Distribuição Aleatória , Receptor ErbB-4 , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
One procedure to treat stenotic coronary arteries is the percutaneous transluminal coronary angioplasty (PTCA). In recent years, drug-eluting stents (DESs) have demonstrated elaborate ways to improve outcomes of intravascular interventions. To enhance DESs, the idea has evolved to design stents that elute specific small interfering RNA (siRNA) for better vascular wall regeneration. Layer-by-layer (LbL) technology offers the possibility of incorporating siRNA nanoplexes (NPs) to achieve bioactive medical implant coatings. The LbL technique was used to achieve hyaluronic acid/chitosan (HA/Chi) films with incorporated Chi-siRNA NPs. The multilayer growth was monitored by quartz crystal microbalance. The coating on the stents and its thickness were analyzed using fluorescence and scanning electron microscopy. All stents showed a homogeneous coating, and the polyelectrolyte multilayers (PEMs) were not disrupted after ethylene oxide sterilization or expansion. The in vitro uptake of fluorescent-labeled NPs from PEMs in primary human endothelial cells (ECs) was analyzed by flow cytometry for 2, 6 and 9 days. Furthermore, stents coated with HA/Chi and Chi-siRNA NPs were expanded into porcine arteries and showed ex vivo delivery of NPs. The films showed no critical results in terms of hemocompatibility. This study demonstrates that Chi-siRNA NPs can be incorporated into PEMs consisting of HA and Chi. We conclude that the NPs were delivered to ECs under in vitro conditions. Furthermore, under ex vivo conditions, NPs were transferred into porcine artery walls. Due to their good hemocompatibility, they might make an innovative tool for achieving bioactive coatings for coronary stents.
Assuntos
Materiais Biocompatíveis , Vasos Coronários , RNA Interferente Pequeno/administração & dosagem , Stents , Endotélio Vascular/citologia , Citometria de Fluxo , Humanos , Microscopia Eletrônica de Varredura , Microscopia de FluorescênciaRESUMO
BACKGROUND: Upregulation of the proto-oncogene plasminogen activator inhibitor-1 (PAI-1) is a common hallmark of various solid tumours, but the mechanisms controlling its expression are not fully understood. METHODS: We investigate microRNAs (miRNAs) regulating PAI-1 in a panel of normal bladder urothelial biopsies, superficial Ta bladder tumours and invasive T1-T4 tumours using expression microarrays and qRT-PCR. The prognostic implications of PAI-1 deregulation are established by tissue microarray staining of non-muscle-invasive bladder tumours. MicroRNA repression of PAI-1 is assayed by ectopic miRNA expression, argonaute immunoprecipitation and luciferase assays. RESULTS: We found that the miR-143/-145 cluster is downregulated in all stages of bladder cancer and inversely correlated with PAI-1 expression. Mature miR-143 and miR-145 are coordinately expressed, and both directly target the PAI-1 3'UTR, leading to reduced PAI-1 mRNA and protein levels. Furthermore, we show that PAI-1 and miR-145 levels may serve as useful prognostic markers for non-muscle-invasive bladder tumours for which accurate progressive outcome is currently difficult to predict. CONCLUSION: This report provides the first evidence for direct miRNA regulation of PAI-1 in bladder cancer. We also demonstrate mRNA co-targeting by a cluster of non-family miRNAs, and suggest miR-145 and PAI-1 as clinically relevant biomarkers in bladder cancer.
Assuntos
Biomarcadores Tumorais/fisiologia , MicroRNAs/fisiologia , Família Multigênica , Inibidor 1 de Ativador de Plasminogênio/fisiologia , Neoplasias da Bexiga Urinária/metabolismo , Regiões 3' não Traduzidas , Sequência de Bases , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Primers do DNA , Humanos , Imuno-Histoquímica , Imunoprecipitação , Hibridização In Situ , MicroRNAs/genética , Análise de Sequência com Séries de Oligonucleotídeos , Inibidor 1 de Ativador de Plasminogênio/genética , Prognóstico , Proto-Oncogene Mas , RNA Mensageiro/genética , Neoplasias da Bexiga Urinária/patologiaRESUMO
G-rich telomeric DNA sequences can form G-quadruplex structures. The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and a shortened derivative (UP1) are active in telomere length regulation, and it has been reported that UP1 can unwind G-quadruplex structures. Here, we investigate the interaction of hnRNP A1 with G-quadruplex DNA structures containing the human telomere repeat (TTAGGG) by gel retardation assays, ensemble fluorescence energy transfer (FRET) spectroscopy, and single molecule FRET microscopy. Our biochemical experiments show that hnRNP A1 binds well to the G-quadruplex telomeric DNA. Ensemble and single molecule FRET measurements provide further insight into molecular conformation: the telomeric DNA overhang is found to be in a folded state in the absence of hnRNP A1 and to remain predominantly in a compact state when complexed with hnRNP A1. This finding is in contrast to the previously reported crystal structures of UP1-telomere DNA complexes where the DNA oligo within the protein-DNA complex is in a fully open conformation.
Assuntos
DNA/química , DNA/metabolismo , Quadruplex G , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Telômero/química , Sequência de Bases , DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Transferência Ressonante de Energia de Fluorescência , Quadruplex G/efeitos dos fármacos , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/farmacologia , Ligação ProteicaRESUMO
Downregulation of miR-145 in a variety of cancers suggests a possible tumor suppressor function for this microRNA. Here, we show that miR-145 expression is reduced in bladder cancer and urothelial carcinoma in situ, compared with normal urothelium, using transcription profiling and in situ hybridization. Ectopic expression of miR-145 induced extensive apoptosis in urothelial carcinoma cell lines (T24 and SW780) as characterized by caspase activation, nuclear condensation and fragmentation, cellular shrinkage, and detachment. However, cell death also proceeded upon caspase inhibition by the pharmacological inhibitor zVAD-fmk and ectopic expression of anti-apoptotic Bcl-2, indicating the activation of an alternative caspase-independent death pathway. Microarray analysis of transcript levels in T24 cells, before the onset of cell death, showed destabilization of mRNAs enriched for miR-145 7mer target sites. Among these, direct targeting of CBFB, PPP3CA, and CLINT1 was confirmed by a luciferase reporter assay. Notably, a 22-gene signature targeted on enforced miR-145 expression in T24 cells was significantly (P<0.00003) upregulated in 55 Ta bladder tumors with concomitant reduction of miR-145. Our data indicate that reduction in miR-145 expression may provide bladder cancer cells with a selective advantage by inhibition of cell death otherwise triggered in malignant cells.
Assuntos
Caspases/metabolismo , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/patologia , Sequência de Bases , Inibidores de Caspase , Morte Celular/genética , Linhagem Celular Tumoral , Biologia Computacional , Regulação para Baixo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de Tempo , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismoRESUMO
A low-density, porous chitosan/poly-(dl-lactide-co-glycolide) (PLGA) microparticle composite scaffold was produced by thermally induced phase separation followed by lyophilization, to provide a bicontinuous microstructure potentially suitable for tissue engineering and locally controlled drug release. PLGA particles were mixed into the chitosan solution and subsequent phase separation during chitosan solidification forced PLGA particles into chitosan phase (Plateau borders). The distributions of volume, surface area, and elongation of 15,422 inclusions of agglomerated PLGA particles were calculated and approximated with log-normal distribution functions from nanotomography reconstructions. Cluster analysis revealed a homogenous inclusion distribution throughout the scaffold. The spatial location and orientation of individual inclusions within the Plateau borders of the scaffold were determined and from these the nearest-neighbor inter-inclusion distance distribution calculated, showing a mean of 2.5 microm. The depth of the inclusions in Plateau borders peaks at 700 or 125 nm, respectively, indicating a step-wise drug release from inclusions successively exposed during scaffold decomposition. Particle diameter ranged from 400 nm to 3 microm and inclusion Feret lengths ranged from 800 nm to 12 microm. These findings on composite morphology and distribution of inclusions are fundamental for predicting scaffold deterioration and particle-mediated drug release during ex vivo and in vivo cell cultivation.
Assuntos
Materiais Biocompatíveis/análise , Materiais Biocompatíveis/química , Alicerces Teciduais/química , Absorciometria de Fóton , Quitosana/química , Análise por Conglomerados , Ácido Láctico/química , Teste de Materiais/métodos , Microscopia Eletrônica de Varredura , Tamanho da Partícula , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Propriedades de Superfície , Engenharia Tecidual/métodosAssuntos
Carcinoma in Situ/genética , Carcinoma in Situ/ultraestrutura , Proteínas de Ciclo Celular/biossíntese , Fator de Transcrição E2F1/biossíntese , Células Germinativas/ultraestrutura , MicroRNAs/biossíntese , RNA Mensageiro/biossíntese , Animais , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patologia , Fator de Transcrição E2F1/genética , Regulação da Expressão Gênica/genética , Células Germinativas/metabolismo , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , MicroRNAs/genética , Biossíntese de Proteínas/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , Testículo/citologia , Testículo/metabolismo , Testículo/ultraestruturaRESUMO
Antisense agents are powerful tools to inhibit gene expression in a sequence-specific manner. They are used for functional genomics, as diagnostic tools and for therapeutic purposes. Three classes of antisense agents can be distinguished by their mode of action: single-stranded antisense oligodeoxynucleotides; catalytic active RNA/DNA such as ribozymes, DNA- or locked nucleic acid (LNA)zymes; and small interfering RNA molecules known as siRNA. The selection of target sites in highly structured RNA molecules is crucial for their successful application. This is a difficult task, since RNA is assembled into nucleoprotein complexes and forms stable secondary structures in vivo, rendering most of the molecule inaccessible to intermolecular base pairing with complementary nucleic acids. In this review, we discuss several selection strategies to identify potential target sites in RNA molecules. In particular, we focus on combinatorial library approaches that allow high throughput screening of sequences for the design of antisense agents.
Assuntos
RNA/uso terapêutico , Animais , Biblioteca Gênica , Humanos , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , RNA/química , RNA Catalítico/química , RNA Catalítico/genética , RNA Catalítico/uso terapêutico , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêuticoRESUMO
A microfabricated cantilever with an internal piezoresistive component has been sensitized with thiol tethered ss-DNA strands and utilized for an in situ, label-free, highly specific, and rapid DNA detection assay. The generation of a differential surface stress onto the functionalized cantilever surface upon target recognition has allowed nanomechanical identification of 12-nucleotide complementary DNA probes with single base mismatch discrimination (sensitivity of 0.2 microM). Interestingly, utilization of an overhang extension distal to the surface enhanced the sensitivity to the 0.01 microM level. The cantilever was functionalized by inkjet printing technology. Replacing the capture probe with locked nucleic acid (LNA) resulted in a faster target probe capture kinetics compared to DNA-DNA hybridization. The capabilities of the piezoresistive cantilever indicate future ergonomic convenience via miniaturization alternative to the conventional laser-based detection method for portable on-site applications.
Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , DNA/análise , Nanoestruturas/química , Sequência de Bases , DNA/química , Microscopia Eletrônica de Varredura , Sondas de Ácido Nucleico/química , Oligonucleotídeos/química , Compostos de Sulfidrila/químicaRESUMO
Predicting RNA secondary structure using evolutionary history can be carried out by using an alignment of related RNA sequences with conserved structure. Accurately determining evolutionary substitution rates for base pairs and single stranded nucleotides is a concern for methods based on this type of approach. Determining these rates can be hard to do reliably without a large and accurate initial alignment, which ideally also has structural annotation. Hence, one must often apply rates extracted from other RNA families with trusted alignments and structures. Here, we investigate this problem by applying rates derived from tRNA and rRNA to the prediction of the much more rapidly evolving 5'-region of HIV-1. We find that the HIV-1 prediction is in agreement with experimental data, even though the relative evolutionary rate between A and G is significantly increased, both in stem and loop regions. In addition we obtained an alignment of the 5' HIV-1 region that is more consistent with the structure than that currently in the database. We added randomized noise to the original values of the rates to investigate the stability of predictions to rate matrix deviations. We find that changes within a fairly large range still produce reliable predictions and conclude that using rates from a limited set of RNA sequences is valid over a broader range of sequences.
Assuntos
Evolução Molecular , Conformação de Ácido Nucleico , RNA/química , Algoritmos , Pareamento de Bases/genética , Bases de Dados de Ácidos Nucleicos , HIV-1/química , HIV-1/genética , Cinética , Modelos Genéticos , Mutação Puntual/genética , RNA/genética , RNA Ribossômico/química , RNA Ribossômico/genética , RNA de Transferência/química , RNA de Transferência/genética , RNA Viral/química , RNA Viral/genética , Alinhamento de Sequência/métodosRESUMO
RNA editing of specific residues by adenosine deamination is a nuclear process catalyzed by adenosine deaminases acting on RNA (ADAR). Different promoters in the ADAR1 gene give rise to two forms of the protein: a constitutive promoter expresses a transcript encoding (c)ADAR1, and an interferon-induced promoter expresses a transcript encoding an N-terminally extended form, (i)ADAR1. Here we show that (c)ADAR1 is primarily nuclear whereas (i)ADAR1 encompasses a functional nuclear export signal in the N-terminal part and is a nucleocytoplasmic shuttle protein. Mutation of the nuclear export signal or treatment with the CRM1-specific drug leptomycin B induces nuclear accumulation of (i)ADAR1 fused to the green fluorescent protein and increases the nuclear editing activity. In concurrence, CRM1 and RanGTP interact specifically with the (i)ADAR1 nuclear export signal to form a tripartite export complex in vitro. Furthermore, our data imply that nuclear import of (i)ADAR1 is mediated by at least two nuclear localization sequences. These results suggest that the nuclear editing activity of (i)ADAR1 is modulated by nuclear export.
Assuntos
Adenosina Desaminase/metabolismo , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Carioferinas/metabolismo , Receptores Citoplasmáticos e Nucleares , Transporte Ativo do Núcleo Celular , Adenosina Desaminase/genética , Animais , Sítios de Ligação , Sequência Conservada , Citoplasma/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Ácidos Graxos Insaturados/farmacologia , Humanos , Carioferinas/antagonistas & inibidores , Camundongos , Sinais de Localização Nuclear , Filogenia , Edição de RNA , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Proteína ran de Ligação ao GTP/metabolismo , Proteína Exportina 1RESUMO
The generation of >30 different HIV-1 mRNAs is achieved by alternative splicing of one primary transcript. The removal of the second tat intron is regulated by a combination of a suboptimal 3' splice site and cis-acting splicing enhancers and silencers. Here we show that hnRNP A1 inhibits splicing of this intron via a novel heterogeneous nuclear ribonucleoprotein (hnRNP) A1-responsive intron splicing silencer (ISS) that can function independently of the previously characterized exon splicing silencer (ESS3). Surprisingly, depletion of hnRNP A1 from the nuclear extract (NE) enables splicing to proceed in NE that contains 100-fold reduced concentrations of U2AF and normal levels of SR proteins, conditions that do not support processing of other efficiently spliced pre-mRNAs. Reconstituting the extract with recombinant hnRNP A1 protein restores splicing inhibition at a step subsequent to U2AF binding, mainly at the time of U2 snRNP association. hnRNP A1 interacts specifically with the ISS sequence, which overlaps with one of three alternative branch point sequences, pointing to a model where the entry of U2 snRNP is physically blocked by hnRNP A1 binding.
Assuntos
Processamento Alternativo/fisiologia , Regulação da Expressão Gênica , Produtos do Gene tat/fisiologia , Inativação Gênica , HIV-1/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , Íntrons/genética , Proteínas Nucleares , Ribonucleoproteínas/fisiologia , Sequência de Bases , Sistema Livre de Células , Elementos Facilitadores Genéticos , HIV-1/genética , Células HeLa , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Ligação Proteica , Subunidades Proteicas , Precursores de RNA/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Viral/biossíntese , RNA Viral/genética , Proteínas Recombinantes de Fusão/fisiologia , Sequências Reguladoras de Ácido Nucleico , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Ribonucleoproteínas/metabolismo , Spliceossomos/metabolismo , Fator de Processamento U2AF , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Splicing of a single HIV-1 primary transcript into more than 30 different mRNAs is regulated by a combination of suboptimal splice sites, cis-acting RNA splicing enhancers and silencers, and trans-acting factors. We have studied the splicing of the second tat intron (SD4 to SA7) and find that activation of splicing by SF2/ASF is mediated by a degenerate exon splicing enhancer (ESE3), consisting of at least three functionally independent sub-elements. One of these sub-elements appears to have both enhancing and silencing properties, depending on the context. SF2/ASF stimulates U2AF65 binding to the suboptimal tat polypyrimidine tract in an ESE3-dependent manner, whereas the exon splicing silencer (ESS3) that is located downstream of the ESE3 inhibits this step. Truncated SF2/ASF protein without the RS domain binds specifically to the ESE3 and retains almost full capacity to stimulate U2AF65 binding and activate splicing. This suggests that SF2/ASF can stimulate the recruitment of U2AF65 by an RS domain-independent mechanism.
Assuntos
Éxons/genética , Produtos do Gene tat/genética , HIV-1/genética , Proteínas Nucleares/metabolismo , Splicing de RNA/genética , Sequências Reguladoras de Ácido Nucleico/genética , Ribonucleoproteínas/metabolismo , Sequência de Bases , Inativação Gênica , Íntrons/genética , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Fatores de Processamento de Serina-Arginina , Fator de Processamento U2AF , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
The untranslated leader of retroviral RNA genomes encodes multiple structural signals that are critical for virus replication. In the human immunodeficiency virus, type 1 (HIV-1) leader, a hairpin structure with a palindrome-containing loop is termed the dimer initiation site (DIS), because it triggers in vitro RNA dimerization through base pairing of the loop-exposed palindromes (kissing loops). Controversy remains regarding the region responsible for HIV-2 RNA dimerization. Different studies have suggested the involvement of the transactivation region, the primer binding site, and a hairpin structure that is the equivalent of the HIV-1 DIS hairpin. We have performed a detailed mutational analysis of the HIV-2 leader RNA, and we also used antisense oligonucleotides to probe the regions involved in dimerization. Our results unequivocally demonstrate that the DIS hairpin is the main determinant for HIV-2 RNA dimerization. The 6-mer palindrome sequence in the DIS loop is essential for dimer formation. Although the sequence can be replaced by other 6-mer palindromes, motifs that form more than two A/U base pairs do not dimerize efficiently. The inability to form stable kissing-loop complexes precludes formation of dimers with more extended base pairing. Structure probing of the DIS hairpin in the context of the complete HIV-2 leader RNA suggests a 5-base pair elongation of the DIS stem as it is proposed in current RNA secondary structure models. This structure is supported by phylogenetic analysis of leader RNA sequences from different viral isolates, indicating that RNA genome dimerization occurs by a similar mechanism for all members of the human and simian immunodeficiency viruses.
Assuntos
Genoma Viral , HIV-2/genética , RNA Viral/química , Sequência de Bases , Primers do DNA , Dimerização , Sondas Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , FilogeniaRESUMO
HIV-1 env expression from certain subgenomic vectors requires the viral regulatory protein Rev, its target sequence RRE, and a 5' splice site upstream of the env open reading frame. To determine the role of this splice site in the 5'-splice-site-dependent Rev-mediated env gene expression, we have subjected the HIV-1 5' splice site, SD4, to a mutational analysis and have analyzed the effect of those mutations on env expression. The results demonstrate that the overall strength of hydrogen bonding between the 5' splice site, SD4, and the free 5' end of the U1 snRNA correlates with env expression efficiency, as long as env expression is suboptimal, and that a continuous stretch of 14 hydrogen bonds can lead to full env expression, as a result of stabilizing the pre-mRNA. The U1 snRNA-mediated stabilization is independent of functional splicing, as a mismatch in position +1 of the 5' splice site that led to loss of detectable amounts of spliced transcripts did not preclude stabilization and expression of the unspliced env mRNA, provided that Rev enables its nuclear export. The nucleotides capable of participating in U1 snRNA:pre-mRNA interaction include positions -3 to +8 of the 5' splice site and all 11 nt constituting the single-stranded 5' end of U1 snRNA. Moreover, env gene expression is significantly decreased upon the introduction of point mutations in several upstream GAR nucleotide motifs, which are mediating SF2/ASF responsiveness in an in vitro splicing assay. This suggests that the GAR sequences may play a role in stabilizing the pre-mRNA by sequestering U1 snRNP to SD4.
Assuntos
Produtos do Gene env/genética , HIV-1/genética , Precursores de RNA/metabolismo , Splicing de RNA , RNA Nuclear Pequeno/metabolismo , RNA Viral/metabolismo , Pareamento Incorreto de Bases , Pareamento de Bases , Sequência de Bases , Ligação de Hidrogênio , Dados de Sequência Molecular , Mutação , Proteínas Nucleares , Conformação de Ácido Nucleico , Estabilidade de RNA , Proteínas de Ligação a RNA , Fatores de Processamento de Serina-ArgininaRESUMO
We isolated a revertant virus after prolonged culturing of a replication-impaired human immunodeficiency virus type 1 (HIV-1) mutant of which the Rev open reading frame was inactivated by mutation of the AUG translation initiation codon. Sequencing of the tat-rev region of this revertant virus identified a second-site mutation in tat that restored virus replication in the mutant background. This mutation activated a cryptic 5' splice site (ss) that, when used in conjunction with the regular HIV 3' ss #5, fuses the tat and rev reading frames to encode a novel T-Rev fusion protein that rescues Rev function. We also demonstrate an alternative route to indirectly activate this cryptic 5' ss by mutational inactivation of an adjacent exon splicing silencer element.