RESUMEN
We previously identified RBPMS as a master regulator of alternative splicing in differentiated smooth muscle cells (SMCs). RBPMS is transcriptionally downregulated during SMC dedifferentiation, but we hypothesized that RBPMS protein activity might be acutely downregulated by post-translational modifications. Publicly available phosphoproteomic datasets reveal that Thr113 and Thr118 immediately adjacent to the RRM domain are commonly both phosphorylated. An RBPMS T113/118 phosphomimetic T/E mutant showed decreased splicing regulatory activity both in transfected cells and in a cell-free in vitro assay, while a non-phosphorylatable T/A mutant retained full activity. Loss of splicing activity was associated with a modest reduction in RNA affinity but significantly reduced RNA binding in nuclear extract. A lower degree of oligomerization of the T/E mutant might cause lower avidity of multivalent RNA binding. However, NMR analysis also revealed that the T113/118E peptide acts as an RNA mimic which can loop back and antagonize RNA-binding by the RRM domain. Finally, we identified ERK2 as the most likely kinase responsible for phosphorylation at Thr113 and Thr118. Collectively, our data identify a potential mechanism for rapid modulation of the SMC splicing program in response to external signals during the vascular injury response and atherogenesis.
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Miocitos del Músculo Liso , Empalme del ARN , Fosforilación , Miocitos del Músculo Liso/metabolismo , Músculo Liso/metabolismo , ARN/metabolismo , Células CultivadasRESUMEN
We have demonstrated previously that the cellular HuR protein binds U-rich elements in the 3' untranslated region (UTR) of Sindbis virus RNA and relocalizes from the nucleus to the cytoplasm upon Sindbis virus infection in 293T cells. In this study, we show that two alphaviruses, Ross River virus and Chikungunya virus, lack the conserved high-affinity U-rich HuR binding element in their 3' UTRs but still maintain the ability to interact with HuR with nanomolar affinities through alternative binding elements. The relocalization of HuR protein occurs during Sindbis infection of multiple mammalian cell types as well as during infections with three other alphaviruses. Interestingly, the relocalization of HuR is not a general cellular reaction to viral infection, as HuR protein remained largely nuclear during infections with dengue and measles virus. Relocalization of HuR in a Sindbis infection required viral gene expression, was independent of the presence of a high-affinity U-rich HuR binding site in the 3' UTR of the virus, and was associated with an alteration in the phosphorylation state of HuR. Sindbis virus-induced HuR relocalization was mechanistically distinct from the movement of HuR observed during a cellular stress response, as there was no accumulation of caspase-mediated HuR cleavage products. Collectively, these data indicate that virus-induced HuR relocalization to the cytoplasm is specific to alphavirus infections and is associated with distinct posttranslational modifications of this RNA-binding protein.
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Infecciones por Alphavirus/metabolismo , Alphavirus/metabolismo , Citoplasma/metabolismo , Proteínas ELAV/metabolismo , Procesamiento Proteico-Postraduccional , Regiones no Traducidas 3'/fisiología , Alphavirus/genética , Infecciones por Alphavirus/genética , Animales , Caspasas/genética , Caspasas/metabolismo , Chlorocebus aethiops , Citoplasma/genética , Citoplasma/virología , Proteínas ELAV/genética , Regulación Viral de la Expresión Génica/fisiología , Células HEK293 , Humanos , Fosforilación/genética , Transporte de Proteínas/genética , Proteolisis , ARN Viral/genética , ARN Viral/metabolismo , Células VeroRESUMEN
INTRODUCTION: Per- and polyfluoroalkyl substances (PFAS) are a class of synthetic compounds used industrially for a wide variety of applications. These PFAS compounds are very stable and persist in the environment. The PFAS contamination is a growing health issue as these compounds have been reported to impact human health and have been detected in both domestic and global water sources. Contaminated water found on military bases poses a potentially serious health concern for active duty military, their families, and the surrounding communities. Previous detection methods for PFAS in contaminated water samples require expensive and time-consuming testing protocols that limit the ability to detect this important global pollutant. The main objective of this work was to develop a novel detection system that utilizes a biological reporter and engineered bacteria as a way to rapidly and efficiently detect PFAS contamination. MATERIALS AND METHODS: The United States Air Force Academy International Genetically Engineered Machine team is genetically engineering Rhodococcus jostii strain RHA1 to contain novel DNA sequences composed of a propane 2-monooxygenase alpha (prmA) promoter and monomeric red fluorescent protein (mRFP). The prmA promoter is activated in the presence of PFAS and transcribes the mRFP reporter. RESULTS: The recombinant R. jostii containing the prmA promoter and mRFP reporter respond to exposure of PFAS by activating gene expression of the mRFP. At 100 µM of perfluorooctanoic acid, the mRFP expression was increased 3-fold (qRT-PCR). Rhodococcus jostii without exposure to PFAS compounds had no mRFP expression. CONCLUSIONS: This novel detection system represents a synthetic biology approach to more efficiently detect PFAS in contaminated samples. With further refinement and modifications, a similar system could be readily deployed in the field around the world to detect this critical pollutant.
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Biología Sintética , Agua Potable , Fluorocarburos/análisis , Humanos , Rhodococcus/genéticaRESUMEN
Synthetic biology encompasses some of the greatest advancements in biology. With improvements in molecular methods and techniques that allow targeted and highly efficient genome manipulation, the capabilities of engineering biology have significantly increased. These enhancements in biotechnology represent significant potential benefits and risks to the global population. It is important that future leaders are trained and understand the incredible benefits, opportunities and risks associated with synthetic biology. The US Department of Defense (DoD) has issued a technical assessment on the future opportunities of synthetic biology and has encouraged the military institutions to expand and encourage bioengineering research programs. At the US Air Force Academy (USAFA), opportunities are provided for future Air Force officers to recognize the potential and risks associated with synthetic biology by participating in the USAFA Synthetic Biology Education Program (USBEP). Cadets can enroll in synthetic biology courses to learn and master molecular biology techniques and work on independent undergraduate research projects. In addition, cadets have the opportunity to join the USAFA's International Genetically Engineered Machine (iGEM) team and compete in the international synthetic biology competition. This report includes details on how USAFA has recruited, enrolled and encouraged synthetic biology research and education among future leaders in the US Air Force.
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Genome-wide linkage analysis using microsatellite markers has been successful in the identification of numerous Mendelian and complex disease loci. The recent availability of high-density single-nucleotide polymorphism (SNP) maps provides a potentially more powerful option. Using the simulated and Collaborative Study on the Genetics of Alcoholism (COGA) datasets from the Genetics Analysis Workshop 14 (GAW14), we examined how altering the density of SNP marker sets impacted the overall information content, the power to detect trait loci, and the number of false positive results. For the simulated data we used SNP maps with density of 0.3 cM, 1 cM, 2 cM, and 3 cM. For the COGA data we combined the marker sets from Illumina and Affymetrix to create a map with average density of 0.25 cM and then, using a sub-sample of these markers, created maps with density of 0.3 cM, 0.6 cM, 1 cM, 2 cM, and 3 cM. For each marker set, multipoint linkage analysis using MERLIN was performed for both dominant and recessive traits derived from marker loci. Our results showed that information content increased with increased map density. For the homogeneous, completely penetrant traits we created, there was only a modest difference in ability to detect trait loci. Additionally, as map density increased there was only a slight increase in the number of false positive results when there was linkage disequilibrium (LD) between markers. The presence of LD between markers may have led to an increased number of false positive regions but no clear relationship between regions of high LD and locations of false positive linkage signals was observed.
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Mapeo Cromosómico , Polimorfismo de Nucleótido Simple/genética , Sitios de Carácter Cuantitativo/genética , Alcoholismo/genética , Simulación por Computador , Bases de Datos Genéticas , Reacciones Falso Positivas , Humanos , Estadísticas no ParamétricasRESUMEN
The impact of RNA viruses on the posttranscriptional regulation of cellular gene expression is unclear. Sindbis virus causes a dramatic relocalization of the cellular HuR protein from the nucleus to the cytoplasm in infected cells. This is to the result of the expression of large amounts of viral RNAs that contain high-affinity HuR binding sites in their 3' UTRs effectively serving as a sponge for the HuR protein. Sequestration of HuR by Sindbis virus is associated with destabilization of cellular mRNAs that normally bind HuR and rely on it to regulate their expression. Furthermore, significant changes can be observed in nuclear alternative polyadenylation and splicing events on cellular pre-mRNAs as a result of sequestration of HuR protein by the 3' UTR of transcripts of this cytoplasmic RNA virus. These studies suggest a molecular mechanism of virus-host interaction that probably has a significant impact on virus replication, cytopathology, and pathogenesis.
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Regiones no Traducidas 3'/genética , Proteínas ELAV/metabolismo , Estabilidad del ARN/genética , ARN Mensajero/química , ARN Viral/genética , Empalme Alternativo/genética , Animales , Sitios de Unión/genética , Línea Celular , Núcleo Celular/genética , Cricetinae , Citoplasma/genética , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Poliadenilación/genética , Procesamiento Postranscripcional del ARN/genética , ARN Viral/biosíntesis , Virus Sindbis/genética , Replicación Viral/genéticaRESUMEN
The cellular mRNA decay machinery plays a major role in regulating the quality and quantity of gene expression in cells. This machinery involves multiple enzymes and pathways that converge to promote the exonucleolytic decay of mRNAs. The transcripts made by RNA viruses are susceptible to degradation by this machinery and, in fact, can be actively targeted. Thus, to maintain gene expression and replication, RNA viruses have evolved a number of strategies to avoid and/or inactivate aspects of the cellular mRNA decay machinery. Recent work uncovering the mechanisms used by RNA viruses to maintain the stability of their transcripts is described below.
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Estabilidad del ARN , Virus ARN/metabolismo , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Animales , Fenómenos Fisiológicos Celulares , Exorribonucleasas/metabolismo , Humanos , Virus ARN/genética , ARN Mensajero/química , ARN Mensajero/genética , ARN Viral/química , ARN Viral/genéticaRESUMEN
Identifying the genetic variants that increase the risk of type 2 diabetes (T2D) in humans has been a formidable challenge. Adopting a genome-wide association strategy, we genotyped 1161 Finnish T2D cases and 1174 Finnish normal glucose-tolerant (NGT) controls with >315,000 single-nucleotide polymorphisms (SNPs) and imputed genotypes for an additional >2 million autosomal SNPs. We carried out association analysis with these SNPs to identify genetic variants that predispose to T2D, compared our T2D association results with the results of two similar studies, and genotyped 80 SNPs in an additional 1215 Finnish T2D cases and 1258 Finnish NGT controls. We identify T2D-associated variants in an intergenic region of chromosome 11p12, contribute to the identification of T2D-associated variants near the genes IGF2BP2 and CDKAL1 and the region of CDKN2A and CDKN2B, and confirm that variants near TCF7L2, SLC30A8, HHEX, FTO, PPARG, and KCNJ11 are associated with T2D risk. This brings the number of T2D loci now confidently identified to at least 10.