Levels Of Reporter Gene Expression Research Articles

Optimization of the 5ʹ untranslated region of mRNA vaccines

To investigate the impact of different 5ʹ untranslated regions (UTRs) on mRNA vaccine translation efficiency, five dual-reporter gene expression plasmids with different 5ʹUTRs were constructed. The corresponding mRNA transcripts were transcribed and capped in vitro. By comparing the expression levels of reporter genes with different 5ʹUTRs, we identified the 5ʹUTR associated with the highest expression level. Subsequently, HIVgp145 mRNA vaccines containing various 5ʹUTRs were constructed and verified. The results demonstrated that mRNA 3 (β-globin 5ʹUTR) displayed the greatest number of green fluorescence-positive cells and the highest luciferase fluorescence intensity in the reporter gene expression system. Further, among the HIVgp145 mRNA vaccines with different 5ʹUTRs, mRNA 7 (β-globin 5ʹUTR) exhibited the highest level of expression. These findings indicate that it is feasible to use the 5ʹUTR of β-globin in an mRNA vaccine, laying the foundation for animal immunogenicity testing.

Transcription Factor Binding Site in Promoter Determines the Pattern of Plasmid-Based Transgene Expression In Vivo.

Understanding the regulation of transgene expression is critical for the success of plasmid-based gene therapy and vaccine development. In this study, we used two sets of plasmid vectors containing secreted embryonic alkaline phosphatase or the mouse IL-10 gene as a reporter and investigated the role of promoter elements in regulating transgene expression in vivo. We demonstrated in mice that hydrodynamic transfer of plasmids with the CMV promoter resulted in a high level of reporter gene expression that declined rapidly over time. In contrast, when plasmids with albumin promoters were used, a lower but sustained gene expression pattern was observed. We also found that plasmids containing a shorter CMV promoter sequence with fewer transcription factor binding sites showed a decrease in the peak level of gene expression without changing the overall pattern of reporter gene expression. The replacement of regulatory elements in the CMV promoter with a single regulatory element of the albumin promoter changed the pattern of transient gene expression seen in the CMV promoter to a pattern of sustained gene expression identical to that of a full albumin promoter. ChIP analyses demonstrated an elevated binding of acetylated histones and TATA box-binding protein to the promoter carrying regulatory elements of the albumin promoter. These results suggest that the strength of a promoter is determined by the number of appropriate transcription factor binding sites, while gene expression persistence is determined by the presence of regulatory elements capable of recruiting epigenetic modifying complexes that make the promoter accessible for transcription. This study provides important insights into the mechanisms underlying gene expression regulation in vivo, which can be used to improve plasmid-based gene therapy and vaccine development.

Genetic regions affecting the replication and pathogenicity of dengue virus type 2.

Dengue is a mosquito-borne disease that has spread to over 100 countries. Its symptoms vary from the relatively mild acute febrile illness called dengue fever to the much more severe dengue shock syndrome. Dengue is caused by dengue virus (DENV), which belongs to the Flavivirus genus of the family Flaviviridae. There are four serotypes of DENV, i.e., DENV1 to DENV4, and each serotype is divided into distinct genotypes. Thailand is an endemic area where all four serotypes of DENV co-circulate. Genome sequencing of the DENV2 that was isolated in Thailand in 2016 and 2017 revealed the emergence of the Cosmopolitan genotype and its co-circulation with the Asian-I genotype. However, it was unclear whether different genotypes have different levels of viral replication and pathogenicity. Focus-forming assay (FFA) results showed that clinical isolates of these genotypes differed in focus size and proliferative capacity. Using circular polymerase extension reaction, we generated parental and chimeric viruses with swapped genes between these two DENV2 genotypes, and compared their focus sizes and infectivity titers using FFA. The results showed that the focus size was larger when the structural proteins and/or non-structural NS1-NS2B proteins were derived from the Cosmopolitan virus. The infectious titers were consistent with the focus sizes. Single-round infectious particle assay results confirmed that chimeric viruses with Cosmopolitan type structural proteins, particularly prM/E, had significantly increased luciferase activity. Replicon assay results showed that Cosmopolitan NS1-NS2B proteins had increased reporter gene expression levels. Furthermore, in interferon-receptor knock-out mice, viruses with Cosmopolitan structural and NS1-NS2B proteins had higher titers in the blood, and caused critical disease courses. These results suggested that differences in the sequences within the structural and NS1-NS2B proteins may be responsible for the differences in replication, pathogenicity, and infectivity between the Asian-I and Cosmopolitan viruses.

식물의 뿌리와 하배축의 옥신 분포와 굴중성 반응에 관한 연구

Plant gravitropic response, regulated by the plant hormone auxin, shows opposite directions in roots and stems. In this study, we examined how the auxin distribution in roots and hypocotyls changes by gravistimulation in Arabidopsis. Auxin distribution was tracked by DR5-GFP fluorescence under gravitropic experimental conditions, designed for the gravitropism analysis in both roots and hypocotyls of Arabidopsis. When the seedlings were subjected to gravistimulation, auxin mainly accumulated in the lower sections of both roots and hypocotyls. Especially in the lower section of hypocotyls, the treatment group’s fl uorescence intensity increased by more than twice compared to that of the control group. We also measured the overall fluorescence intensity in the entire hypocotyl region, and found to increase by 40% in the treatment group compared to the control group. These fi ndings suggest that the absolute amount of auxin in the hypocotyls increased by gravistimulation. To confirm the increase in auxin concentration, we quantitatively checked the expression of DR5-GUS reporter gene, which is another marker for auxin levels. The results confi rmed that the reporter gene expression level increased significantly, particularly in the gravistimulated hypocotyls. Taken together, this study suggests that gravistimulation not only causes the redistribution of auxin but also overall increase in the concentration of auxin in Arabidopsis.

Validating a Promoter Library for Application in Plasmid-Based Diatom Genetic Engineering.

While diatoms are promising synthetic biology platforms, there currently exists a limited number of validated genetic regulatory parts available for genetic engineering. The standard method for diatom transformation, nonspecific introduction of DNA into chromosomes via biolistic particle bombardment, is low throughput and suffers from clonal variability and epigenetic effects. Recent developments in diatom engineering have demonstrated that autonomously replicating episomal plasmids serve as stable expression platforms for diverse gene expression technologies. These plasmids are delivered via bacterial conjugation and, when combined with modular DNA assembly technologies, provide a flexibility and speed not possible with biolistic-mediated strain generation. In order to expand the current toolbox for plasmid-based engineering in the diatom Phaeodactylum tricornutum, a conjugation-based forward genetics screen for promoter discovery was developed, and application to a diatom genomic DNA library defined 252 P. tricornutum promoter elements. From this library, 40 promoter/terminator pairs were delivered via conjugation on episomal plasmids, characterized in vivo, and ranked across 4 orders of magnitude difference in reporter gene expression levels.

Optimizing a Novel Fate-Mapping Strategy to Assess Tissue Resident Macrophages in Muscle Regeneration

Tissue resident macrophages (TRMs) are heterogenous innate immune cells involved in tissue homeostasis and repair, yet their role in muscle regeneration is currently unknown. TRMs differ from classical monocyte-derived macrophages (MDMs) in both ontogeny and function. Our inability to differentiate TRMs from MDMs has limited the development of subset specific immunotherapies. The novel Cx3cr1CreER/+:RosaTd/+ fate-mapping mouse model employs tamoxifen inducible labeling of MDMs and TRMs with a TdTomato reporter (Td) that persists only in self-renewing TRMs, while MDMs are replaced by Td- monocyte progenitors over a 4-week period. This strategy has been successfully used in adult mice heart and brain tissue. However, in the skeletal muscle, expression level of the Cx3cr1 reporter gene is unknown. We assessed muscle TRM Cx3cr1 expression in neonate (P3), young (P21), and adult (10wk) mice to optimize a pulse/chase strategy to label muscle TRMs with the Td reporter. Like heart and brain tissue, we found muscle TRMs express high levels of Cx3cr1 throughout development and adulthood suggesting viability of this strategy. We performed a single injection of tamoxifen in adult Cx3cr1CreER/+:RosaTd/+ mice and showed ~20% induction in blood monocytes and muscle TRMs. Although Td induction was poor, levels were similar between the blood and muscle, suggesting accuracy of the model. The next step is to optimize tamoxifen administration to achieve >90% labelling in blood and muscle. To assess spatial localization of TRMs with muscle resident stem cells (satellite cells) we optimized immunofluorescence staining on tissue sections. We found a statistically significant increase in colocalization of macrophages with satellite cells in healthy and injured gastrocnemius tissue, consistent with their predicted role in satellite cell proliferation. Once complete, our fate-mapping approach will allow for the labelling of muscle TRM subsets and reveal their role in the regeneration process.

Muscle Specific Promotors for Gene Therapy - A Comparative Study in Proliferating and Differentiated Cells.

Depending on the therapy approach and disease background, the heterogeneity of muscular tissues complicates the development of targeted gene therapy, where either expression in all muscle types or restriction to only one muscle type is warranted. Muscle specificity can be achieved using promotors mediating tissue specific and sustained physiological expression in the desired muscle types but limited activity in non-targeted tissue. Several muscle specific promotors have been described, but direct comparisons between them are lacking. Here we present a direct comparison of muscle specific Desmin-, MHCK7, microRNA206- and Calpain3 promotor. To directly compare these muscle specific promotors we utilized transfection of reporter plasmids using an in vitro model based on electrical pulse stimulation (EPS) to provoke sarcomere formation in 2D cell culture for quantification of promotor activities in far differentiated mouse and human myotubes. We found that Desmin- and MHCK7 promotors showed stronger reporter gene expression levels in proliferating and differentiated myogenic cell lines than miR206 and CAPN3 promotor. However, Desmin and MHCK7 promotor promoted gene expression also cardiac cells whereas miR206 and CAPN3 promotor expression was restricted to skeletal muscle. Our results provides direct comparison of muscle specific promotors with regard to expression strengths and specificity as this is important feature to avoid undesired transgene expression in non-target muscle cells for a desired therapy approach.

Characterizing the tumor selectivity of oncolytic adenovirus ICVB-1042: Analysis of S-phase induction, viral replication, and cytolytic activity in human primary normal cell types.

e14503 Background: Adenoviral-based oncolytic virus (OV) therapeutics include mutations that enable activity in tumor cells and limit toxicity in normal cells. These tumor selectivity approaches often involve mutating viral E1A function or expression; however, such modifications allow viral activity in normal cells or limit anti-tumor potency. ICVB-1042 was engineered with dual modifications in E1A and E4orf6/7, resulting in superior tumor selectivity that does not compromise lytic potency in tumor cells. This improved tumor selective virus minimizes off-tumor activity and promotes robust tumor lysis. Methods: ICVB-1042 was compared to viruses with wildtype E1A and E4orf6/7 and a clinically tested virus with an E1A mutation (ICVB-2006). Viral function and killing were assessed in primary normal human renal (HRE) and bronchial (BrE) epithelial cells in the presence of the inhibitor palbociclib to pharmacologically block S-phase entry via host cell signaling. HRE and BrE were infected with virus and evaluated for indicators of viral toxicity in normal cells. Viral replication was quantified by droplet digital polymerase chain reaction (ddPCR); cell lysis was quantified by WST-1 assay. An S-phase induction assay was developed using an mCherry-Geminin reporter that allows imaging-based quantification of viral-dependent entry to S-phase. Viral activity in cells was compared with and without the CDK4/6 inhibitor using a viral-encoded fluorescent reporter to determine the requirement for host cell proliferation on viral function. Results: ICVB-1042 demonstrated superior protection in normal cells to comparator ICVB-2006 and viruses lacking tumor selectivity modifications. Palbociclib steeply reduced viral reporter gene expression (63-88% reduction), viral replication ( > 10-fold reduction), and cytolytic activity of ICVB-1042, while non-selective viruses were unaffected by this treatment. Non-selective virus infection resulted in 3.3- to 6.1-fold higher levels of S-phase entry compared to ICVB-1042. ICVB-2006 infection resembled non-selective viruses at the level of S-phase induction (3.1- to 3.3-fold above ICVB-1042) and viral reporter gene expression was modestly inhibited by palbociclib (12-31% reduction). Conclusions: The improved tumor selectivity of ICVB-1042 results in protection of normal, non-proliferative cells from viral-mediated toxicity. The conventional approach of ICVB-2006 with a single E1A mutation leaves viral replication and cell death in normal human cells mostly intact. Together, these observations support that ICVB-1042 presents normal cell protection and reduced toxicity risks associated with OV therapy.

Remote control of transgene expression using noninvasive near-infrared irradiation.

This study investigated whether noninvasive near-infrared (NIR) energy could be transduced into heat in deep-seated organs in which adenovirus type-5 vectors tend to accumulate, thereby activating heat shock protein (HSP) promoter-mediated transgene expression, without local administration of photothermal agents. NIR irradiation of the subdiaphragmatic and left dorsocranial part of the abdominal cavity of adult immunocompetent C3H/HeNRj mice with an 808-nm laser effectively increased the temperature of the irradiated regions of the liver and spleen, respectively, resulting in the accumulation of the heat-inducible HSP70 protein. Spatial control of transgene expression was achieved in the NIR-irradiated regions of the mice administered an adenoviral vector carrying a firefly luciferase (fLuc) coding sequence controlled by a human HSP70B promoter, as assessed by bioluminescence and immunohistochemistry analyses. Levels of reporter gene expression were modulated by controlling NIR power density. Spatial control of transgene expression through NIR-focused activation of the HSP70B promoter, as well as temporal regulation by administering rapamycin was achieved in the spleens of mice inoculated with an adenoviral vector encoding a rapamycin-dependent transactivator driven by the HSP70B promoter and an adenoviral vector carrying a fLuc coding sequence controlled by the rapamycin-activated transactivator. Mice that were administered rapamycin and exposed to NIR light expressed fLuc activity in the splenic region, whereas no activity was detected in mice that were only administered rapamycin or vehicle or only NIR-irradiated. Thus, in the absence of any exogenously supplied photothermal material, remote control of heat-induced transgene expression can be achieved in the liver and spleen by means of noninvasive NIR irradiation.

Stable overexpression and targeted gene deletion of the causative agent of ash dieback Hymenoscyphus fraxineus

BackgroundDue to the infection with the invasive ascomycete Hymenoscyphus fraxineus, which has been replacing the closely related and non-pathogenic native Hymenoscyphus albidus, the European ashes, Fraxinus excelsior (also known as the common ash), Fraxinus angustifolia (also known as narrow-leaved ash) and Fraxinus ornus (also known as the manna ash) are at risk. Hymenoscyphus fraxineus is the causative agent of ash dieback of the European ashes, but is non-pathogenic to the native Asian ash Fraxinus mandshurica (also known as the Manchurian ash). Even though the invasion of H. fraxineus is a great threat for ashes in Europe, the fungal biology is still poorly understood. By the use of live cell imaging and targeted gene knock-out, the fungal life cycle and host–pathogen interaction can be studied in more detail.ResultsHere, we developed a protocol for the preparation of protoplasts from mycelium of H. fraxineus, for their regeneration and for stable transformation with reporter genes and targeted gene knock-out by homologous recombination. We obtained mutants with various levels of reporter gene expression which did not correlate with the number of integrations. In an in vitro infection assay, we demonstrated the suitability of reporter gene overexpression for fungal detection in plant tissue after inoculation. As a proof of principle for targeted gene knock-out, the hygromycin resistance cassette of a reporter gene-expressing mutant was replaced with a geneticin resistance cassette.ConclusionsThe invasive fungal pathogen H. fraxineus is threatening the European ashes. To develop strategies for pest management, a better understanding of the fungal life cycle and its host interaction is crucial. Here, we provide a protocol for stable transformation of H. fraxineus to obtain fluorescence reporter strains and targeted gene knock-out mutants. This protocol will help future investigations on the biology of this pathogen.

Use of a Bacterial Artificial Chromosome to Generate Recombinant SARS-CoV-2 Expressing Robust Levels of Reporter Genes.

Reporter-expressing recombinant virus represents an excellent option and a powerful tool to investigate, among others, viral infection, pathogenicity, and transmission, as well as to identify therapeutic compounds that inhibit viral infection and prophylactic vaccines. To combat the ongoing coronavirus disease 2019 (COVID-19) pandemic, we have established a robust bacterial artificial chromosome (BAC)-based reverse genetics (RG) system to rapidly generate recombinant severe acute respiratory syndrome coronavirus 2 (rSARS-CoV-2) to study the contribution of viral proteins in viral pathogenesis. In addition, we have engineered reporter-expressing recombinant viruses in which we placed the reporter genes upstream of the viral nucleocapsid (N) gene to promote high levels of reporter gene expression, which facilitates the study of SARS-CoV-2 in vitro and in vivo. To date, we have shared our BAC-based RG system with more than 100 laboratories around the world, which has helped to expedite investigations with SARS-CoV-2. However, genetic manipulation of the BAC containing the entire SARS-CoV-2 genome (~30,000 nt) is challenging. Herein, we provide the technical details to engineer rSARS-CoV-2 using the BAC-based RG approach. We describe (i) assembly of the full-length (FL) SARS-CoV-2 genome sequences into the empty pBeloBAC, (ii) verification of pBeloBAC-FL, (iii) cloning of a Venus reporter gene into pBeloBAC-FL, and (iv) recovery of the Venus-expressing rSARS-CoV-2. By following this protocol, researchers with knowledge of basic molecular biology and gene engineering techniques will be able to generate wild-type (WT) and reporter-expressing rSARS-CoV-2. IMPORTANCE We have established a bacterial artificial chromosome (BAC)-based RG system to generate recombinant severe acute respiratory syndrome coronavirus 2 (rSARS-CoV-2) and to engineer reporter-expressing recombinant viruses to assess viral infection in vitro and in vivo. To date, we have shared our BAC-based RG system with more than 100 laboratories around the world, which has helped to expedite investigations with SARS-CoV-2. However, genetic manipulation of the BAC containing the full-length SARS-CoV-2 genome of ~30,000 nucleotides is challenging. Here, we provide all the detailed experimental steps required for the successful generation of wild-type (WT) recombinant SARS-CoV-2 (rSARS-CoV-2). Likewise, we provide a comprehensive protocol on how to generate and rescue rSARS-CoV-2 expressing high levels of a Venus fluorescent reporter gene from the locus of the viral nucleocapsid (N) protein. By following these protocols, researchers with basic knowledge in molecular biology will be able to generate WT and Venus-expressing rSARS-CoV-2 within 40 days.

The Pan-ErbB tyrosine kinase inhibitor afatinib inhibits multiple steps of the mammarenavirus life cycle

The mammarenavirus Lassa virus (LASV) causes a life-threatening acute febrile disease, Lassa fever (LF). To date, no US Food and Drug Administration (FDA)-licensed medical countermeasures against LASV are available. This underscores the need for the development of novel anti-LASV drugs. Here, we screen an FDA-approved drug library to identify novel anti-LASV drug candidates using an infectious-free cell line expressing a functional LASV ribonucleoprotein (vRNP), where levels of vRNP-directed reporter gene expression serve as a surrogate for vRNP activity. Our screen identified the pan-ErbB tyrosine kinase inhibitor afatinib as a potent inhibitor of LASV vRNP activity. Afatinib inhibited multiplication of lymphocytic choriomeningitis virus (LCMV) a mammarenavirus closely related to LASV. Cell-based assays revealed that afatinib inhibited multiple steps of the LASV and LCMV life cycles. Afatinib also inhibited multiplication of Junín virus vaccine strain Candid#1, indicating that afatinib can have antiviral activity against a broad range of human pathogenic mammarenaviruses.

Monitoring SARS-CoV-2 Infection Using a Double Reporter-Expressing Virus.

ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the highly contagious agent responsible for the coronavirus disease 2019 (COVID-19) pandemic. An essential requirement for understanding SARS-CoV-2 biology and the impact of antiviral therapeutics is a robust method to detect the presence of the virus in infected cells or animal models. Despite the development and successful generation of recombinant (r)SARS-CoV-2-expressing fluorescent or luciferase reporter genes, knowledge acquired from their use in in vitro assays and/or in live animals is limited to the properties of the fluorescent or luciferase reporter genes. Herein, for the first time, we engineered a replication-competent rSARS-CoV-2 that expresses both fluorescent (mCherry) and luciferase (Nluc) reporter genes (rSARS-CoV-2/mCherry-Nluc) to overcome limitations associated with the use of a single reporter gene. In cultured cells, rSARS-CoV-2/mCherry-Nluc displayed similar viral fitness as rSARS-CoV-2 expressing single reporter fluorescent and luciferase genes (rSARS-CoV-2/mCherry and rSARS-CoV-2/Nluc, respectively) or wild-type (WT) rSARS-CoV-2, while maintaining comparable expression levels of both reporter genes. In vivo, rSARS-CoV-2/mCherry-Nluc has similar pathogenicity in K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mice than rSARS-CoV-2 expressing individual reporter genes or WT rSARS-CoV-2. Importantly, rSARS-CoV-2/mCherry-Nluc facilitates the assessment of viral infection and transmission in golden Syrian hamsters using in vivo imaging systems (IVIS). Altogether, this study demonstrates the feasibility of using this novel bioreporter-expressing rSARS-CoV-2 for the study of SARS-CoV-2 in vitro and in vivo.IMPORTANCE Despite the availability of vaccines and antivirals, the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to ravage health care institutions worldwide. Previously, we generated replication-competent recombinant (r)SARS-CoV-2 expressing fluorescent or luciferase reporter proteins to track viral infection in vitro and/or in vivo. However, these rSARS-CoV-2 are restricted to express only a single fluorescent or a luciferase reporter gene, limiting or preventing their use in specific in vitro assays and/or in vivo studies. To overcome this limitation, we have engineered a rSARS-CoV-2 expressing both fluorescent (mCherry) and luciferase (Nluc) genes and demonstrated its feasibility to study the biology of SARS-CoV-2 in vitro and/or in vivo, including the identification and characterization of neutralizing antibodies and/or antivirals. Using rodent models, we visualized SARS-CoV-2 infection and transmission through in vivo imaging systems (IVIS).

Сравнительный анализ эффективности общедоступных методов трансфекции модельных клеточных линий для задач биотехнологии

Transient gene expression is one of the most common methods in molecular biology, equally relevant for basic research projects and biotechnological industries. Despite the existence of commercial transfection systems, which afford high transfection efficiency and high expression levels of reporter genes, expanding such systems to industrial scales is often problematic due to high costs of the reagents. The well-described methods of cationic and calcium-phosphate transfection are accessible and ensure reproducible results at much lower costs. This study is aimed at comparative validation of calcium phosphate and cationic (polyethylenimine-based) transfection protocols along with the commercially available TurboFect reagent for mono- and cotransfections on a panel of commonly used cell lines including HEK293T, Huh7, BHK-21, CHO and MRC5. The efficiency of transfection with plasmid constructs encoding different fluorescent proteins was measured by flow cytometry. Of all the tested methods, calcium phosphate transfection afforded the highest efficiency of plasmid DNA delivery in all the cell lines except BHK21, for which the PEI method turned out to be more efficient than calcium phosphate transfection, and CHO, for which both methods showed comparable efficiency.

An Erwinia amylovora inducible promoter for improvement of apple fire blight resistance

Key messagepPPO16, the first Ea-inducible promoter cloned from apple, can be a useful component of intragenic strategies to create fire blight resistant apple genotypes.Intragenesis is an important alternative to transgenesis to produce modified plants containing native DNA only. A key point to develop such a strategy is the availability of regulatory sequences controlling the expression of the gene of interest. With the aim of finding apple gene promoters either inducible by the fire blight pathogen Erwinia amylovora (Ea) or moderately constitutive, we focused on polyphenoloxidase genes (PPO). These genes encode oxidative enzymes involved in many physiological processes and have been previously shown to be upregulated during the Ea infection process. We found ten PPO and two PPO-like sequences in the apple genome and characterized the promoters of MdPPO16 (pPPO16) and MdKFDV02 PPO-like (pKFDV02) for their potential as Ea-inducible and low-constitutive regulatory sequences, respectively. Expression levels of reporter genes fused to these promoters and transiently or stably expressed in apple were quantified after various treatments. Unlike pKFDV02 which displayed a variable activity, pPPO16 allowed a fast and strong expression of transgenes in apple following Ea infection in a Type 3 Secretion System dependent manner. Altogether our results does not confirmed pKFDV02 as a constitutive and weak promoter whereas pPPO16, the first Ea-inducible promoter cloned from apple, can be a useful component of intragenic strategies to create fire blight resistant apple genotypes.

Marburg and Ebola Virus mRNA 3' Untranslated Regions Contain Negative Regulators of Translation That Are Modulated by ADAR1 Editing.

ABSTRACTThe filovirus family includes deadly pathogens such as Ebola virus (EBOV) and Marburg virus (MARV). A substantial portion of filovirus genomes encode 5′ and 3′ untranslated regions (UTRs) of viral mRNAs. Select viral genomic RNA sequences corresponding to 3′ UTRs are prone to editing by adenosine deaminase acting on RNA 1 (ADAR1). A reporter mRNA approach, in which different 5′ or 3′ UTRs were inserted into luciferase-encoding mRNAs, demonstrates that MARV 3′ UTRs yield different levels of reporter gene expression, suggesting modulation of translation. The modulation occurs in cells unable to produce microRNAs (miRNAs) and can be recapitulated in a MARV minigenome assay. Deletion mutants identified negative regulatory regions at the ends of the MARV nucleoprotein (NP) and large protein (L) 3′ UTRs. Apparent ADAR1 editing mutants were previously identified within the MARV NP 3′ UTR. Introduction of these changes into the MARV nucleoprotein (NP) 3′ UTR or deletion of the region targeted for editing enhances translation, as indicated by reporter assays and polysome analysis. In addition, the parental NP 3′ UTR, but not the edited or deletion mutant NP 3′ UTRs, induces a type I interferon (IFN) response upon transfection into cells. Because some EBOV isolates from the West Africa outbreak exhibited ADAR1 editing of the viral protein of 40 kDa (VP40) 3′ UTR, VP40 3′ UTRs with parental and edited sequences were similarly assayed. The EBOV VP40 3′ UTR edits also enhanced translation, but neither the wild-type nor the edited 3′ UTRs induced IFN. These findings implicate filoviral mRNA 3′ UTRs as negative regulators of translation that can be inactivated by innate immune responses that induce ADAR1.IMPORTANCE UTRs comprise a large percentage of filovirus genomes and are apparent targets of editing by ADAR1, an enzyme with pro- and antiviral activities. However, the functional significance of the UTRs and ADAR1 editing has been uncertain. This study demonstrates that MARV and EBOV 3′ UTRs can modulate translation, in some cases negatively. ADAR1 editing or deletion of select regions within the translation suppressing 3′ UTRs relieves the negative effects of the UTRs. These data indicate that filovirus 3′ UTRs contain translation regulatory elements that are modulated by activation of ADAR1, suggesting a complex interplay between filovirus gene expression and innate immunity.

Construction of DNA Tools for Hyperexpression in Marchantia Chloroplasts.

Chloroplasts are attractive platforms for synthetic biology applications since they are capable of driving very high levels of transgene expression, if mRNA production and stability are properly regulated. However, plastid transformation is a slow process and currently limited to a few plant species. The liverwort Marchantia polymorpha is a simple model plant that allows rapid transformation studies; however, its potential for protein hyperexpression has not been fully exploited. This is partially due to the fact that chloroplast post-transcriptional regulation is poorly characterized in this plant. We have mapped patterns of transcription in Marchantia chloroplasts. Furthermore, we have obtained and compared sequences from 51 bryophyte species and identified putative sites for pentatricopeptide repeat protein binding that are thought to play important roles in mRNA stabilization. Candidate binding sites were tested for their ability to confer high levels of reporter gene expression in Marchantia chloroplasts, and levels of protein production and effects on growth were measured in homoplastic transformed plants. We have produced novel DNA tools for protein hyperexpression in this facile plant system that is a test-bed for chloroplast engineering.

Mutation of OUR1/OsbZIP1, which encodes a member of the basic leucine zipper transcription factor family, promotes root development in rice through repressing auxin signaling

A well-developed root system is essential for efficient water uptake, particularly in drought-prone environments. However, the molecular mechanisms underlying the promotion of root development are poorly understood. We identified and characterized a rice mutant, outstanding rooting1 (our1), which exhibited a well-developed root system. The our1 mutant displayed typical auxin-related phenotypes, including elongated seminal root and defective gravitropism. Seminal root elongation in the our1 mutant was accelerated via the promotion of cell division and elongation. In addition, compared with the wild type, the density of short and thin lateral roots (S-type LRs) was reduced in the our1 mutant, whereas that of long and thick LRs (L-type LRs) was increased. Expression of OUR1, which encodes OsbZIP1, a member of the basic leucine zipper transcription factor family, was observed in the seminal root tip and sites of LR emergence, wherein attenuation of reporter gene expression levels controlled by the auxin response promoter DR5 was also observed in the our1 mutant. Taken together, our results indicate that the our1 gene promotes root development by suppressing auxin signaling, which may be a key factor contributing to an improvement in root architecture.

TAEL 2.0: An Improved Optogenetic Expression System for Zebrafish.

Inducible gene expression systems are valuable tools for studying biological processes. We previously developed an optogenetic gene expression system called TAEL that is optimized for use in zebrafish. When illuminated with blue light, TAEL transcription factors dimerize and activate gene expression downstream of the TAEL-responsive C120 promoter. By using light as the inducing agent, the TAEL/C120 system overcomes limitations of traditional inducible expression systems by enabling fine spatial and temporal regulation of gene expression. In this study, we describe ongoing efforts to improve the TAEL/C120 system. We made modifications to both the TAEL transcriptional activator and the C120 regulatory element, collectively referred to as TAEL 2.0. We demonstrate that TAEL 2.0 consistently induces higher levels of reporter gene expression and at a faster rate, but with comparable background and toxicity as the original TAEL system. With these improvements, we were able to create functional stable transgenic lines to express the TAEL 2.0 transcription factor either ubiquitously or with a tissue-specific promoter. We demonstrate that the ubiquitous line in particular can be used to induce expression at late embryonic and larval stages, addressing a major deficiency of the original TAEL system. This improved optogenetic expression system will be a broadly useful resource for the zebrafish community.

Fine gene expression regulation by minor sequence variations downstream of the polyadenylation signal.

The termination of transcription is a complex process that substantially contributes to gene regulation in eukaryotes. Previously, it was noted that a single cytosine deletion at the position + 32bp relative to the single polyadenylation signal AAUAAA (hereafter the dC mutation) causes a 2-fold increase in the transcription level of the upstream eGFP reporter in mouse embryonic stem cells. Here, we analyzed the conservation of this phenomenon in immortalized mouse, human and drosophila cell lines and the influence of the dC mutation on the choice of the pre-mRNA cleavage sites. We have constructed dual-reporter plasmids to accurately measure the effect of the dC and other nearby located mutations on eGFP mRNA level by RT-qPCR. In this way, we found that the dC mutation leads to a 2-fold increase in the expression level of the upstream eGFP reporter gene in cultured mouse and human, but not in drosophila cells. In addition, 3' RACE analysis demonstrated that eGFP pre-mRNAs are cut at multiple positions between + 14 to + 31, and that the most proximal cleavage site becomes almost exclusively utilized in the presence of the dC mutation. We alsoidentified new short sequence variations located within positions + 25.. + 40 and + 33.. + 48 that increase eGFP expression up to ~2-4-fold. Altogether, the positive effect of the dC mutation seems to be conserved in mouse embryonic stem cells, mouse embryonic 3T3 fibroblasts and human HEK293T cells. In the latter cells, the dC mutation appears to be involved in regulating pre-mRNA cleavage site selection. Finally, a multiplexed approach is proposed to identify motifs located downstream of cleavage site(s) that are essential for transcription termination.