Minimal pre-mRNA substrates with natural and converted sites for full-round U insertion and U deletion RNA editing in trypanosomes

Trypanosome RNA editing by uridylate insertion or deletion cycles is a mitochondrial mRNA maturation process catalyzed by multisubunit complexes. A full-round of editing entails three consecutive steps directed by partially complementary guide RNAs: pre-mRNA cleavage, U addition or removal, and ligation. The structural and functional composition of editing complexes is intensively studied, but their molecular interactions in and around editing sites are not completely understood. In this study, we performed a systematic analysis of distal RNA requirements for full-round insertion and deletion by purified editosomes. We define minimal substrates for efficient editing of A6 and CYb model transcripts, and established a new substrate, RPS12. Important differences were observed in the composition of substrates for insertion and deletion. Furthermore, we also showed for the first time that natural sites can be artificially converted in both directions: from deletion to insertion or from insertion to deletion. Our site conversions enabled a direct comparison of the two editing kinds at common sites during substrate minimization and demonstrate that all basic determinants directing the editosome to carry out full-round insertion or deletion reside within each editing site. Surprisingly, we were able to engineer a deletion site into CYb, which exclusively undergoes insertion in nature

CITOTOXICIDAD Y ACTIVIDAD ANTIVIRAL DE EXTRACTOS DE CHILES (Capsicum spp), CYTOTOXICITY AND ANTIVIRAL ACTIVITY OF PEPPER EXTRACTS (Capsicum spp)

RESUMENEn este trabajo se evaluaron  la citotoxicidad y la actividad antiviral en contra del virus herpes simplex tipo 1 (VHS-1)  de extractos  de chile Jalapeño, Serrano, Guajillo, Ancho, Pimiento (Capsicum annuum L. var. annuum.) y Habanero (Capsicum chinense Jacq), así como de los fenilpropanoides, ácidos fenólicos y flavonoides puros que fueron identificados por HPLC-MS en los extractos  de chile. La concentración que causa el 50 % de toxicidad en células Vero fue determinada (CC50) así como el potencial antiviral de los extractos de chiles y de los compuestos puros,  expresado como la concentración inhibitoria del 50 %  (CI50) en contra de los efectos citopáticos en células Vero infectadas con el virus. El índice de selectividad fue calculado como la relación de CC50 entreCI50.  El extracto de Pimiento mostró las más baja citotoxicidad (CC50= 9.82 ± 0.06 mg/mL) y la más alta  actividad antiviral (IC50= 0.56 ± 0.02 mg/mL), con el mayor índice de selectividad de 17.5. Los extractos de chile Ancho y Guajillo mostraron también una alta capacidad antiviral. El extracto de chile Habanero presentó la mayor citotoxicidad y el menor índice de selectividad. Los compuestos fenólicos contenidos en los extractos también mostraron actividad antiviral, lo que sugiere que pueden ser estos compuestos los responsables de la actividad antiviral de los chiles.Palabras clave: Capsicum spp, citotoxicidad, actividad antiviral, compuestos fenólicos.

Binding of hnRNP H and U2AF65 to Respective G-codes and a Poly-Uridine Tract Collaborate in the N50-5'ss Selection of the REST N Exon in H69 Cells

The splicing of the N exon in the pre-mRNA coding for the RE1-silencing transcription factor (REST) results in a truncated protein that modifies the expression pattern of some of its target genes. A weak 3’ss, three alternative 5’ss (N4-, N50-, and N62-5’ss) and a variety of putative target sites for splicing regulatory proteins are found around the N exon; two GGGG codes (G2-G3) and a poly-Uridine tract (N-PU) are found in front of the N50-5’ss. In this work we analyzed some of the regulatory factors and elements involved in the preferred selection of the N50-5’ss (N50 activation) in the small cell lung cancer cell line H69. Wild type and mutant N exon/b-globin minigenes recapitulated N50 exon splicing in H69 cells, and showed that the N-PU and the G2-G3 elements are required for N50 exon splicing. Biochemical and knockdown experiments identified these elements as U2AF65 and hnRNP H targets, respectively, and that they are also required for N50 exon activation. Compared to normal MRC5 cells, and in keeping with N50 exon activation, U2AF65, hnRNP H and other splicing factors were highly expressed in H69 cells. CLIP experiments revealed that hnRNP H RNA-binding occurs first and is a prerequisite for U2AF65 RNA binding, and EMSA and CLIP experiments suggest that U2AF65-RNA recognition displaces hnRNP H and helps to recruit other splicing factors (at least U1 70K) to the N50-5’ss. Our results evidenced novel hnRNP H and U2AF65 functions: respectively, U2AF65-recruiting to a 5’ss in humans and the hnRNP H-displacing function from two juxtaposed GGGG codes

NF90 Binds the Dengue Virus RNA 3′ Terminus and is a Positive Regulator of Dengue Virus Replication

Background Viral RNA translation and replication are regulated by sequence and structural elements in the 5′ and 3′ untranslated regions (UTR) and by host cell and/or viral proteins that bind them. Dengue virus has a single-stranded RNA genome with positive polarity, a 5′ m7GpppG cap, and a conserved 3′-terminal stem loop (SL) that is linked to proposed functions in viral RNA transcription and translation. Mechanisms explaining the contributions of host proteins to viral RNA translation and replication are poorly defined, yet understanding host protein-viral RNA interactions may identify new targets for therapeutic intervention. This study was directed at identifying functionally significant host proteins that bind the conserved dengue virus RNA 3′ terminus. Methodology/Principal Findings Proteins eluted from a dengue 3′ SL RNA affinity column at increasing ionic strength included two with double-strand RNA binding motifs (NF90/DRBP76 and DEAH box polypeptide 9/RNA helicase A (RHA)), in addition to NF45, which forms a heterodimer with NF90. Although detectable NF90 and RHA proteins localized to the nucleus of uninfected cells, immunofluorescence revealed cytoplasmic NF90 in dengue virus-infected cells, leading us to hypothesize that NF90 has a functional role(s) in dengue infections. Cells depleted of NF90 were used to quantify viral RNA transcript levels and production of infectious dengue virus. NF90 depletion was accompanied by a 50%-70% decrease in dengue RNA levels and in production of infectious viral progeny. Conclusions/Significance The results indicate that NF90 interacts with the 3′ SL structure of the dengue RNA and is a positive regulator of dengue virus replication. NF90 depletion diminished the production of infectious dengue virus by more than 50%, which may have important significance for identifying therapeutic targets to limit a virus that threatens more than a billion people worldwide.Ruth L. Kirschstein National Research Service Award (NIH-NRSA GM64985)UNCF-Merck Postdoctoral FellowshipNational Institute of Allergy and Infectious Diseases (U.S.)Ellison Medical Foundatio

Dengue-2 Structural Proteins Associate with Human Proteins to Produce a Coagulation and Innate Immune Response Biased Interactome

<p>Abstract</p> <p>Background</p> <p>Dengue virus infection is a public health threat to hundreds of millions of individuals in the tropical regions of the globe. Although Dengue infection usually manifests itself in its mildest, though often debilitating clinical form, dengue fever, life-threatening complications commonly arise in the form of hemorrhagic shock and encephalitis. The etiological basis for the virus-induced pathology in general, and the different clinical manifestations in particular, are not well understood. We reasoned that a detailed knowledge of the global biological processes affected by virus entry into a cell might help shed new light on this long-standing problem.</p> <p>Methods</p> <p>A bacterial two-hybrid screen using DENV2 structural proteins as bait was performed, and the results were used to feed a manually curated, global dengue-human protein interaction network. Gene ontology and pathway enrichment, along with network topology and microarray meta-analysis, were used to generate hypothesis regarding dengue disease biology.</p> <p>Results</p> <p>Combining bioinformatic tools with two-hybrid technology, we screened human cDNA libraries to catalogue proteins physically interacting with the DENV2 virus structural proteins, Env, cap and PrM. We identified 31 interacting human proteins representing distinct biological processes that are closely related to the major clinical diagnostic feature of dengue infection: haemostatic imbalance. In addition, we found dengue-binding human proteins involved with additional key aspects, previously described as fundamental for virus entry into cells and the innate immune response to infection. Construction of a DENV2-human global protein interaction network revealed interesting biological properties suggested by simple network topology analysis.</p> <p>Conclusions</p> <p>Our experimental strategy revealed that dengue structural proteins interact with human protein targets involved in the maintenance of blood coagulation and innate anti-viral response processes, and predicts that the interaction of dengue proteins with a proposed human protein interaction network produces a modified biological outcome that may be behind the hallmark pathologies of dengue infection.</p

Congreso Internacional de Responsabilidad Social Apuestas para el desarrollo regional.

Congreso Internacional de Responsabilidad Social: apuestas para el desarrollo regional [Edición 1 / Nov. 6 - 7: 2019 Bogotá D.C.]El Congreso Internacional de Responsabilidad Social “Apuestas para el Desarrollo Regional”, se llevó a cabo los días 6 y 7 de noviembre de 2019 en la ciudad de Bogotá D.C. como un evento académico e investigativo liderado por la Corporación Universitaria Minuto de Dios -UNIMINUTO – Rectoría Cundinamarca cuya pretensión fue el fomento de nuevos paradigmas, la divulgación de conocimiento renovado en torno a la Responsabilidad Social; finalidad adoptada institucionalmente como postura ética y política que impacta la docencia, la investigación y la proyección social, y cuyo propósito central es la promoción de una “sensibilización consciente y crítica ante las situaciones problemáticas, tanto de las comunidades como del país, al igual que la adquisición de unas competencias orientadas a la promoción y al compromiso con el desarrollo humano y social integral”. (UNIMINUTO, 2014). Dicha postura, de conciencia crítica y sensibilización social, sumada a la experiencia adquirida mediante el trabajo articulado con otras instituciones de índole académico y de forma directa con las comunidades, permitió establecer como objetivo central del evento la reflexión de los diferentes grupos de interés, la gestión de sus impactos como elementos puntuales que contribuyeron en la audiencia a la toma de conciencia frente al papel que se debe asumir a favor de la responsabilidad social como aporte seguro al desarrollo regional y a su vez al fortalecimiento de los Objetivos de Desarrollo Sostenible

Congreso Internacional de Responsabilidad Social Apuestas para el desarrollo regional.

Congreso Internacional de Responsabilidad Social: apuestas para el desarrollo regional [Edición 1 / Nov. 6 - 7: 2019 Bogotá D.C.]El Congreso Internacional de Responsabilidad Social “Apuestas para el Desarrollo Regional”, se llevó a cabo los días 6 y 7 de noviembre de 2019 en la ciudad de Bogotá D.C. como un evento académico e investigativo liderado por la Corporación Universitaria Minuto de Dios -UNIMINUTO – Rectoría Cundinamarca cuya pretensión fue el fomento de nuevos paradigmas, la divulgación de conocimiento renovado en torno a la Responsabilidad Social; finalidad adoptada institucionalmente como postura ética y política que impacta la docencia, la investigación y la proyección social, y cuyo propósito central es la promoción de una “sensibilización consciente y crítica ante las situaciones problemáticas, tanto de las comunidades como del país, al igual que la adquisición de unas competencias orientadas a la promoción y al compromiso con el desarrollo humano y social integral”. (UNIMINUTO, 2014). Dicha postura, de conciencia crítica y sensibilización social, sumada a la experiencia adquirida mediante el trabajo articulado con otras instituciones de índole académico y de forma directa con las comunidades, permitió establecer como objetivo central del evento la reflexión de los diferentes grupos de interés, la gestión de sus impactos como elementos puntuales que contribuyeron en la audiencia a la toma de conciencia frente al papel que se debe asumir a favor de la responsabilidad social como aporte seguro al desarrollo regional y a su vez al fortalecimiento de los Objetivos de Desarrollo Sostenible

Viral Interference and Persistence in Mosquito-Borne Flaviviruses

Mosquito-borne flaviviruses are important pathogens for humans, and the detection of two or more flaviviruses cocirculating in the same geographic area has often been reported. However, the epidemiological impact remains to be determined. Mosquito-borne flaviviruses are primarily transmitted through Aedes and Culex mosquitoes; these viruses establish a life-long or persistent infection without apparent pathological effects. This establishment requires a balance between virus replication and the antiviral host response. Viral interference is a phenomenon whereby one virus inhibits the replication of other viruses, and this condition is frequently associated with persistent infections. Viral interference and persistent infection are determined by several factors, such as defective interfering particles, competition for cellular factors required for translation/replication, and the host antiviral response. The interaction between two flaviviruses typically results in viral interference, indicating that these viruses share common features during the replicative cycle in the vector. The potential mechanisms involved in these processes are reviewed here

Complete cycles of bloodstream trypanosome RNA editing in vitro

RNA editing in kinetoplastid protists is required for the maturation of mitochondrial pre-mRNAs and occurs by protein-catalyzed cycles of uridylate insertion and deletion. During the complex life cycle of Trypanosoma brucei this process is differentially regulated in the mammalian bloodstream and insect procyclic stages. Complementary guide RNAs (gRNAs) direct editing, but the abundance of these transcripts is not developmentally controlled. The establishment of in vitro systems that recreate efficient RNA editing in bloodstream T. brucei would be valuable for mechanistic studies of regulation. Here we describe a robust in vitro system that reconstitutes full cycles of both U insertion and U deletion in bloodstream trypanosomes, and the first direct comparisons of the in vitro systems for strains of mammalian and insect stages