Effect of encephalomyocarditis virus 3'Untranslated region on GFP transient expression: implications in recombinant protein production / Efecto de la Región 3'-no traducida del virus de la encefalomiocarditis sobre la expresión transitoria de GFP: implicaciones en la producción de proteínas recombinantes

The enrichment of therapeutic protein production yield in mammalian cell cultures by modulating mRNA stability is a fairly new strategy in biotechnological applications. Here, we describe the application of 3'-untranslated region (3'UTR) from RNA viral genome to modulate mRNA stability.The data obtained showed that the use of the 3 'UTR sequence of the encephalomyocarditis virus (EMCV 3'UTR) downstream of the target gene was not able to significantly modulate the free energy density indicators of the RNA. However, the sequence influenced the stability of the mRNA (and, therefore, the amount of protein production) in a cell type and time-dependent manner, indicating a central role of mRNA-stabilizing binding sites/cellular factors in this process. Our data might be of interest for the biotechnology community to improve recombinant protein production in mammalian cell cultures and RNA-based therapy/vaccination approaches.

El enriquecimiento de la producción terapéutica de proteínas en cultivos de células de mamíferos mediante la modulación de la estabilidad del ARNm es una estrategia nueva en aplicaciones biotecnológicas. Se describe la aplicación de la región 3'-no traducida (3'UTR) del genoma viral ARN para modular la estabilidad del ARNm. Los datos obtenidos mostraron que el uso de la secuencia 3'UTR del virus de la encefalomiocarditis (EMCV 3'UTR) aguas abajo del gen objetivo no pudo modular significativamente los indicadores de densidad de energía libre del ARN. Sin embargo, la secuencia influyó en la estabilidad del ARNm (y, por lo tanto, en la cantidad de producción de proteínas) dependiente de la célula y del tiempo, lo que indica un papel central de los sitios de unión estabilizadores de ARNm/factores celulares en este proceso. Nuestros datos podrían ser de interés para la comunidad biotecnológica para mejorar la producción de proteínas recombinantes en cultivos de células de mamíferos y en enfoques de terapia/vacunación basados en ARN.

Characterization of the mRNA untranslated regions [UTR] of the Trypanosoma cruzi LYT1 isoforms derived by alternative trans-splicing / Caracterización de las regiones no traducidas ARN [UTR] de las isoformas de Trypanosoma cruzi LYT1 derivadas de un trans-empalme alternativo / Caracterização das regiões não-traduzidas do RNA [UTR] das isoformas de Trypanosoma cruzi LYT1 derivadas de uma junção trans-alternativa

Abstract In trypanosomatids, gene expression is mainly regulated at posttranscriptional level, through mechanisms based on the interaction between RNA Binding Proteins [RBPs] and motifs present in the untranslated regions [UTRs] of the mRNAs, which altogether form ribonucleoproteic complexes [RNP] that define the fate of the mRNA. The pre-mRNA derived from the LYT1 gene of Trypanosoma cruzi, is processed by alternative trans-splicing, resulting in different mRNAs which code for the isoforms mLYTl and kLYTl, proteins having differential expression, cellular location and function. The aim of this study was to characterize the 5' and 3' UTRs of the LYT1 mRNAs as the initial step towards the objective of identification of the RBPs responsible for their differential expression. The presence of the two types of 5' UTRs were confirmed in two T. cruzi isolates belonging to the DTU I, thus, corroborating the occurrence of alternative trans-splicing also in the LYT1 gene of this T. cruzi DTU. In addition, for the first time, was unscovered the existence of two types of LYT1 mRNAs transcripts, differing in length by 116 nts, that are generated by alternative polyadenylation. Furthermore, an in-silico analysis of the experimentally obtained UTRs, and ten additional LYT1 sequences retrieved from TritrypDB and GenBank databases, together with a thoroughly search of structural motifs, showed a remarkable conservation of relevant structural motifs previously associated with RNA metabolism in the different UTRs; these elements might be involved in the differential stage-specific expression of each LYT1 isoform.

Resumen En los trypanosomátidos, la expresión génica se regula principalmente en el nivel post-transcripcional mediante mecanismos basados en la interacción entre las proteínas de unión del ARN [RBP] y las figuras presentes en las regiones no traducidas [UTR] de las ARN, que en conjunto forman complejos ribonucleoproteicos [RNP] que definen el destino de la ARN. El pre-ARN derivado del gen LYT1 del Trypanosoma cruzi es procesado por trans-empalme alternativo, dando como resultado diferentes ARN que codifican las isoformas mLYTl y kLYTl, proteínas con expresión diferencial, localización celular y función. El objetivo de este estudio fue caracterizar los 5' y 3' UTR de las ARN LYT1 como el paso inicial hacia la identificación de los RPB responsables de la expresión diferencial. Se confirmó la presencia de los dos tipos de 5' UTR en dos aislantes del T. cruzi pertenecientes al DTU I; de esta forma también se comprobó la ocurrencia del trans-empalme alternativo en el gen LYT1 de este T. cruzi DTU. Además, por primera vez, se pudo demostrar la existencia de dos tipos de transcripciones de ARN LYT1, que difieren en longitud por 116 nts, y son generadas por poliadenilación alternativa. Adicionalmente, se realizó un análisis in-silico de la UTR obtenida experimentalmente, y otras diez secuencias LYT1 recuperadas de las bases de datos TritrypDB y GenBank, junto con una búsqueda exhaustiva de figuras estructuradas, mostrando una notable conservación de los figuras estructurales asociadas con el metabolismo del ARN en los diferentes UTR; estos elementos podrían estar implicados en la expresión diferenciada de la etapa específica de cada isoforma LYT1.

Resumo Nos tripanossomatídeos, a expressão génica é regulada principalmente a nível pós-transcricional mediante mecanismos baseados na interação entre as proteínas de união do RNA [RBPs] e as fugiras presentes nas regiões não-traduzidas [UTRs] do RNA. O pré-RNA derivado do gene LYT1 do Trypanosoma cruzí é processado por uma junção trans-alternativa, resultando em diferentes RNA que codificam as isoformas mLYTl e kLYTl, proteínas com expressão, localização celular e função diferenciadas. O objetivo de este estudo foi caracterizar as 5' e 3' UTRs dos RNAs LYT1 como sendo o passo inicial na identificação das RBPs responsáveis pela expressão diferenciada. A presença dos dois tipos de 5' UTRs foi confirmada em dois isolados de T. cruzí pertencentes ao DTU I; corroborando assim com a ocorrência da junção trans-alternativa no gene LYT1 de este T. crují DTU. Adicionalmente, se demonstrou pela primeira vez a existência de dois tipos de transcrições de RNA LYT1, que se diferenciam em comprimento por 116 nts, e são geradas por poliadenização alternativa. Além disso, realizou-se uma análise in-sílico da UTR obtida experimentalmente e outras dez sequencias LYT1 recuperadas das bases de dados TritrypDB e GenBank, junto com uma busca exaustiva de figuras estruturadas, mostrando uma notável conservação das figuras estruturais associadas com o metabolismo do RNA nas diferentes UTRs. Estes elementos poderiam estar envolvidos na expressão estágio-específica diferenciada de cada isoforma LYT1.

ADAR1-mediated RNA-editing of 3'UTRs in breast cancer

BACKGROUND: Whole transcriptome RNA variant analyses have shown that adenosine deaminases acting on RNA ( ADAR ) enzymes modify a large proportion of cellular RNAs, contributing to transcriptome diversity and cancer evolution. Despite the advances in the understanding of ADAR function in breast cancer, ADAR RNA editing functional consequences are not fully addressed. RESULTS: We characterized A to G(I) mRNA editing in 81 breast cell lines, showing increased editing at 3'UTR and exonic regions in breast cancer cells compared to immortalized non-malignant cell lines. In addition, tumors from the BRCA TCGA cohort show a 24% increase in editing over normal breast samples when looking at 571 well-characterized UTRs targeted by ADAR1. Basal-like subtype breast cancer patients with high level of ADAR1 mRNA expression shows a worse clinical outcome and increased editing in their 3'UTRs. Interestingly, editing was particularly increased in the 3'UTRs of ATM, GINS4 and POLH transcripts in tumors, which correlated with their mRNA expression. We confirmed the role of ADAR1 in this regulation using a shRNA in a breast cancer cell line (ZR-75-1). CONCLUSIONS: Altogether, these results revealed a significant association between the mRNA editing in genes related to cancer-relevant pathways and clinical outcomes, suggesting an important role of ADAR1 expression and function in breast cancer.

miR-483-5p promotes prostate cancer cell proliferation and invasion by targeting RBM5

ABSTRACT Objective: miR-483-5p has been identified as a miRNA oncogene in certain cancers. However, its role in prostate cancer has not been sufficiently investigated. In this study, we investigated the role of miR-483-5p in prostate cancer and examined RBM5 regulation by miR-483-5p. Material and methods: Expression levels of miR-483-5p were determined by quantitative real-time PCR. The effect of miR-483-5p on proliferation was evaluated by MTT assay, cell invasion was evaluated by trans-well invasion assays, and target protein expression was determined by western blotting in LNCaP, DU-145, and PC-3 cells. Luciferase reporter plasmids were constructed to confirm the action of miR-483-5p on downstream target gene RBM5 in HEK-293T cells. Results: we observed that miR-483-5p was upregulated in prostate cancer cell lines and tissues. A miR-483-5p inhibitor inhibited prostate cancer cell growth and invasion in DU-145 and PC-3 cells. miR-483-5p directly bound to the 3' untranslated region (3'UTR) of RBM5 in HEK-293T cells. RBM5 overexpression inhibited prostate cancer cell growth and invasion in LNCaP cells. Enforced RBM5 expression alleviated miR-483-5p promotion of prostate cancer cell growth and invasion in LNCaP cells. Conclusion: The present study describes a potential mechanism underlying a miR-483-5p/RBM5 link that contributes to prostate cancer development.

Overexpression of miR-484 and miR-744 in Vero cells alters Dengue virus replication

BACKGROUND Dengue is considered one of the world’s most important mosquito-borne diseases. MicroRNAs (miRNAs) are small non-coding single-stranded RNAs that play an important role in the regulation of gene expression in eukaryotes. Although miRNAs possess antiviral activity against many mammalian-infecting viruses, their involvement in Dengue virus (DENV) replication remains poorly understood. OBJECTIVE To determine the role of miR-484 and miR-744 in DENV infection and to examine whether DENV infection alters the expression of both miRNAs. METHODS We used bioinformatics tools to explore the relationship between DENV and cellular miRNAs. We then overexpressed miR-484 or miR-744 in Vero cells to examine their role in DENV replication using flow cytometry, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), and western blotting. FINDINGS We found several cellular miRNAs that target a conserved region within the 3′ untranslated region (3′ UTR) of the genome of the four DENV serotypes and found that overexpression of miR-484 or miR-744 inhibits infection by DENV-1 to DENV-4. Furthermore, we observed that DENV RNA might be involved in the downregulation of endogenous miR-484 and miR-744. CONCLUSION Our study identifies miR-484 and miR-744 as two possible restriction host factors against DENV infection. However, further studies are needed to directly verify whether miR-484 and miR-744 both have an anti-DENV effect in vivo.

Optimization of one-step real-time PCR for the X-tail target of HCV as a diagnostic test

Infection with hepatitis C virus (HCV) is a global public health issue. The bloodborne nature of HCV transmission poses a substantial risk to healthcare workers, due to occupational exposure to needlestick injuries and blood and other body fluids containing the virus. Undiagnosed HCV infection, including in healthcare workers, represents a growing problem worldwide as the infected population ages, and HCV-related mortality and morbidity is expected to rise substantially over the coming decades. Consequently, diagnostic tests for HCV play an important role in this scenario. The aim of this study was to standardize a one-step RT-PCR assay for detection of HCV. The test demonstrated reproducibility, sensibility (100%), and the limit of detection was set at 100IU/mL. Our study indicates that this assay can be used as a diagnostic tool to follow up healthcare workers after occupational exposure

Estudios de la región 5'UTR del gen FMR-1 en pacientes con falla ovárica prematura / Studies of the 5' - UTRTR region in the FMR-1 gene in patients withe Premature Ovarian Failure

La falla ovárica prematura (FOP) es un síndrome de patogénesis multicausal que afecta aproximadamente al 1% de las mujeres en edad reproductiva. Numerosos estudios asocian el estado de premutación (amplificación del número de tripletes CGG entre 50/55 y 200 repeticiones) en el gen FMR-1 y FOP. Alrededor de un 4% de las pacientes FOP presentan alelos con premutación. La amplificación del número de tripletes por encima de 200 repeticiones causa el Síndrome de Fragilidad del X (SFX). El objetivo del presente trabajo fue estudiar la región 5´ no codificante del gen en un grupo de pacientes FOP de Argentina. La región de interés se amplificó por PCR a partir de muestras de ADN de 100 pacientes FOP y 145 mujeres controles. Los alelos de las pacientes y controles fueron agrupados en 7 categorías de acuerdo al número de tripletes obtenidos. Se observó que el número de repeticiones más frecuente se encuentra en el rango de 26 a 30 tripletes, tanto en pacientes como en controles. En el grupo de pacientes FOP, 5/197 (2.6%) alelos no relacionados estudiados presentaron un número de tripletes CGG mayor a 50, mientras que sólo 1 de 290 (0.34%) para el grupo control. Todas las pacientes FOP con valores de tripletes CGG mayor a 50 presentaron amenorrea secundaria. Estos resultados están en concordancia con lo comunicado para otras poblaciones acerca de la existencia de una asociación entre la premutación del gen FMR-1 y el desarrollo de FOP. Asimismo, los resultados obtenidos refuerzan la importancia de la genotipificación del gen FMR-1 en las pacientes FOP, a los efectos de estimar el riesgo de su descendencia para el SFX.

Premature ovarian failure (POF) is a syndrome of multicausal pathogenesis that affects 1% of women before the age of 40. Several studies associate the premutation state (CGG repeats increased in number between 50/55 and 200) in the FMR-1 gene and POF. About 4% of POF women have alleles in the FMR-1 gene in the permutation range. An increase above 200 in the number of triplets in this gene causes the Fragile X Syndrome (FXS). The purpose of the present study was to analyze the 5´untranslated region of the FMR-1 gene in a group of patients from Argentina. The region of interest was amplified by PCR from DNA samples of 100 POF patients and 145 control women. Alleles from controls and patients were grouped in 7 categories according to the number of triplets obtained. We observed that the most frequent number of repeats ranged from 26 to 30 triplets, in both patient and control groups. In the POF group, 5 out of 197 (2.6%) not related alleles presented a number of CGG triplets higher than 50, while only 1 out of 290 (0.34%) was present in controls. All POF patients with a number of CGG repeats higher than 50 presented secondary amenorrhea. These results are in accordance with previous reports from other populations showing an association between the premutation state in the FMR-1 gene and POF development. In addition, these results reinforce the importance of genotyping POF patients to estimate the risk of their offspring for Fragile X Syndrome.

Torque teno virus (TTV) is prevalent in Brazilian non-human primates and chickens (Gallus gallus domesticus) / Torque teno virus (TTV) es prevalente en primates no humanos brasileños y pollos (Gallus gallus domesticus)

Torque Teno virus (TTV) is an infectious agent of worldwide distribution isolated by the first time as the agent of an acute post-transfusion hepatitis in a patient in Japan. It has been classified into a new floating genus called Anellovirus. Recent studies showed that TTV can also be identified in serum specimens obtained from domesticated farm animals and from non-human primates. To better understand the relationship between TTV and their hosts, a study to detect virus in the serum and whole blood of Brazilian non-human primates and in the plasm of chickens was performed by applying the PCR-UTR-A technique, followed by a genomic sequence and phylogenetic analysis. By nested-PCR-UTR, the DNA of TTV was detected in sera from 4 (5.3 percent) of 75 Cebus apella, 2 (40 percent) of 5 Alouatafusca, 1 (20 percent) of 5 Alouata caraya, 1 (5.2 percent) of 19 Callithrixpenicilata, 1 (4 percent) of 25 Callithrixjacchus, 1 (20 percent) of 5 Saimiri sciureus and 1 (25 percent) of 4 Leontopithecus chrysomelas. Phylogenetic analysis revealed that sequences detected in 8 samples clustered with TTV sequences So-TTV2 (Sagüínus oedipus) and At-TTV3 (Aotes Trivirgatus). Three sequences showed similarity with a human Torque Teno Minivirus (TLMV). TTV ORF2 DNA was detected in one sera sample and one whole blood sample of non-human primates and in one plasm sample of chicken. Phylogenetic analysis revealed that the sequences amplified by the ORF2 region show no difference between human, non-human primates and chicken. This is the first report of TTV in Brazilian new world non-human primates and chicken.

Torque Teno virus (TTV) es una agente infeccioso de distribución mundial, aislado por primera vez como el agente de una hepatitis aguda posterior a la transfusión de un paciente en Japón. Se ha clasificado en un nuevo género flotante llamado Anellovirus. Recientes estudios han demostrado que TTV también puede ser identificado en el suero de especímenes obtenidos desde granjas de animales domésticos y desde primates no humanos. Para entender mejor la relación entre la TTV y sus huéspedes, fue realizado un estudio para detectar el virus en el suero y la sangre de primates no humanos brasileños y en el plasma de pollos mediante la aplicación de la técnica PCR-UTR-A, seguida de una secuencia genómica y análisis filogenético. Por medio de PCR-UTR-anidado, el ADN de TTV fue detectado en sueros de 4 de 75 (5,3 por ciento)Cebus apella, 2 de 5 (40 por ciento) Alouata fusca, 1 de 5 (20 por ciento) de Alouata caraya, 1 de 19 (5,2 por ciento) de Callithrixpenicilata, 1 de 25 (4 por ciento) Callithrixjacchus, 1 de 5 (20 por ciento) de Saimiri sciureus y 1 de 4 (25 por ciento) de Leontopithecus chrysomelas. El análisis filogenético reveló secuencias detectadas en 8 muestras agrupadas con TTV secuencias So-TTV2 (Sagüínus oedipus) y At-TTV3 (Aotes Trivirgatus). Tres secuencias mostraron similitud con el Torque Teno Minivirus humano (TLMV). Fue detectado TTV ORF2 ADN en una muestra de suero y una muestra de sangre de primates no-humanos y en una muestra de plasma de pollo. El análisis filogenético reveló que las secuencias amplificadas por la región ORF2 no muestran ninguna diferencia entre humanos, primates no humanos y pollos. Este es el primer informe de nuevos TTV en primates-no humanos brasileños y en pollos.

The untranslated regions of genes from Trypanosoma cruzi: perspectives for functional characterization of strains and isolates

The sequencing of Trypanosoma cruzi genome has been completed and a great deal of information is now available. However, the organization of protozoa genomes is somewhat elusive and much effort must be applied to reveal all the information coded in the nucleotide sequences. Among the DNA segments that needs further investigation are the untranslated regions of genes. Many of the T. cruzi genes that were revealed by the genome sequencing lack information about the untranslated regions. In this paper, some features of these untranslated segments as well as their applications in T. cruzi populations are discussed.

A microsatellite study of bovine solute carrier family 11 a1 (Slc11a1) gene diversity in Mexico in relation to bovine tuberculosis

Bovine tuberculosis, caused by Mycobacterium bovis, is a disease of socio-economic and public health importance and of significance to international trade regulation. Allelic variants of several genes have been implicated in the genetic susceptibility to tuberculosis in some human populations, but little is known in cattle. We surveyed 34 European, 18 Asian, 20 Creole and 23 hybrid bovines for polymorphisms of the bovine solute carrier family 11 a1(Slc11a1) gene, formerly known as natural resistance associated macrophage protein (Nramp1), gene by typing the cattle using two microsatellite loci closely linked to this gene. The microsatellites used were 311-22, located at the 3' untranslated region (3' UTR) of the Slc11a1 gene, and ARO28 situated about 0.6 cM upstream of the same gene Based on allele size in base pairs (bp) we determined five 311-223 locus variants (221, 223, 225, 227 and 229 bp) and 12 ARO28 loci. There was marked diversity and a very high level of heterozygosity in most of the cattle surveyed except the Europeans bovines and especially Holsteins in relation to the 3' UTR microsatellite locus.

New prevalence estimate of TT virus (TTV) infection in low- and high-risk population from Säo Paulo, Brazil

The prevalence of TT virus (TTV) infection was investigated by Polymerase Chain Reaction (PCR) in low- (blood donors and healthy children/adolescents) and high-risk (hemophiliacs) groups from Säo Paulo, Brazil. Primers based on the untranslated region (UTR) of the viral genome proved to be much more ubiquitous, leading to much higher frequencies for both groups ( > or = 81 percent) than the earlier N22-PCR directed to the open reading frame 1 (blood donors, 5.5 percent, and hemophiliacs, 42.3 percent). The UTR-PCR also revealed an interesting profile for healthy children/adolescents: very high prevalence at the early years and significant decrease in male teenagers. The N22-PCR, in turn, demonstrated higher frequency in hemophiliacs treated with fresh blood products (58 percent), than in those treated with virus-inactivated clotting factors (9.4 percent) and blood donors (5.5 percent)