Green Fluorescent Protein RNA Research Articles

Engineering a replication-incompetent viral vector for the delivery of therapeutic RNA in crustaceans.

Viral disease pandemics are a major cause of economic losses in crustacean farming worldwide. While RNA interference (RNAi)-based therapeutics have shown promise at a laboratory scale, without an effective oral delivery platform, RNA-based therapy will not reach its potential against controlling viral diseases in crustaceans. Using a reverse-engineered shrimp RNA virus, Macrobrachium rosenbergii nodavirus (MrNV), we have developed a shrimp viral vector for delivering an engineered RNA cargo. By replacing the RNA-dependent RNA polymerase (RdRp) protein-coding region of MrNV with a cargo RNA encoding green fluorescent protein (GFP) as a proof-of-concept, we generated a replication-incompetent mutant MrNV(ΔRdRp) carrying the GFP RNA cargo resulting in MrNV(ΔRdRp)-GFP. Upon incorporating MrNV(ΔRdRp)-GFP in the diet of the marine Pacific white shrimp (Penaeus vannamei), MrNV(ΔRdRp) particles were visualized in hemocytes demonstrating successful vector internalization. Fluorescence imaging of hemocytes showed the expression of GFP protein and the MrNV capsid RNA (RNA2) as well as the incorporated GFP RNA cargo. Detection of cargo RNA in hepatopancreas and pleopods indicated the systemic spread of the viral vector. The quantitative load of both the MrNV RNA2 and GFP RNA progressively diminished within 8 days postadministration of the viral vector, which indicated a lack of MrNV(ΔRdRp)-GFP replication in shrimp. In addition, no pathological hallmarks of the wild-type MrNV infection were detected using histopathology in the target tissue of treated shrimp. The data unequivocally demonstrated the successful engineering of a replication-incompetent viral vector for RNA delivery, paving the way for the oral delivery of antiviral therapeutics in farmed crustaceans.

A photoelectron imaging study of the deprotonated GFP chromophore anion and RNA fluorescent tags.

Green fluorescent protein (GFP), together with its family of variants, is the most widely used fluorescent protein for in vivo imaging. Numerous spectroscopic studies of the isolated GFP chromophore have been aimed at understanding the electronic properties of GFP. Here, we build on earlier work [A. V. Bochenkova, C. Mooney, M. A. Parkes, J. Woodhouse, L. Zhang, R. Lewin, J. M. Ward, H. Hailes, L. H. Andersen and H. H. Fielding, Chem. Sci., 2017, 8, 3154] investigating the impact of fluorine and methoxy substituents that have been employed to tune the electronic structure of the GFP chromophore for use as fluorescent RNA tags. We present photoelectron spectra following photoexcitation over a broad range of wavelengths (364-230 nm) together with photoelectron angular distributions following photoexcitation at 364 nm, which are interpreted with the aid of quantum chemistry calculations. The results support the earlier high-level quantum chemistry calculations that predicted how fluorine and methoxy substituents tune the electronic structure and we find evidence to suggest that the methoxy substituents enhance internal conversion, most likely from the 2ππ* state which has predominantly Feshbach resonance character, to the 1ππ* state.

The Citrus leaf blotch virus movement protein acts as silencing suppressor

To counteract plant antiviral defense based on RNA silencing, many viruses express proteins that inhibit this mechanism at different levels. The genome of Citrus leaf blotch virus (CLBV) encodes a 227-kDa protein involved in replication, a 40-kDa movement protein (MP), and a 41-kDa coat protein (CP). To determine if any of these proteins might have RNA silencing suppressor activities, we have used Agrobacterium-mediated transient assays in the green fluorescent protein (GFP)-expressing Nicotiana benthamiana line 16c. Only CLBV MP was able to suppress intracellular GFP silencing induced by expression of either single- or double-stranded (ds) GFP RNA, but not cell-to-cell or long distance spread of the silencing signal. The MP suppressor activity was weak compared to other characterized viral suppressor proteins. Overall our data indicate that MP acts as a suppressor of local silencing probably by interfering in the silencing pathway downstream of the steps of dsRNA and small RNAs generation.

V2 protein encoded by Tomato yellow leaf curl China virus is an RNA silencing suppressor

The V2 protein of Tomato yellow leaf curl China virus (TYLCCNV) was identified as an RNA silencing suppressor by Agrobacterium-mediated co-infiltration. The V2 protein could inhibit local RNA silencing, systemic RNA silencing of the green fluorescent protein (GFP) gene and the spread of a systemic GFP RNA silencing signal. However, the V2 could not interfere with the cell-to-cell spread of RNA silencing. Subcellular localization assay indicated that the V2 protein was distributed in the cytoplasm of Nicotiana benthamiana cells, and accumulated in irregular cytoplasmic bodies. The V2 bound 21nt and 24nt small interfering RNA (siRNA) duplexes and 24nt single-stranded (ss)-siRNA but not 21nt ss-siRNA in electrophoresis mobility shift assays. Expression of the V2 protein via the Potato virus X (PVX) vectors heterogenous system induced severe symptoms in N. benthamiana. In a yeast two-hybrid system, TYLCCNV V2 could interact with itself, but not with SlSGS3, which is known to been involved in RNA silencing pathway and to interact with a closely related Tomato yellow leaf curl virus (TYLCV) V2. These results indicate that TYLCCNV V2 is an RNA silencing suppressor, possibly through sequestering siRNA molecules.

Human dendritic cells expressing hepatitis C virus core protein display transcriptional and functional changes consistent with maturation

Hepatitis C virus (HCV) causes a chronic liver infection, which may result in cirrhosis and hepatocellular carcinoma. Impairment of the maturation process in dendritic cells (DCs) may be one of the mechanisms responsible for immune evasion of HCV. The core and NS3 proteins are among the most conserved HCV proteins and play a key role in viral clearance. To evaluate the effects of these proteins on DCs, monocyte-derived immature DCs (iDCs) were transfected with in vitro transcribed (IVT) HCV core or NS3 RNA and treated with maturation factors. Neither core nor NS3 had an inhibitory effect on DC maturation; however, transfection of iDCs with IVT core RNA appeared to result in changes compatible with maturation. To investigate this in more detail, the transcriptional profiles of iDCs transfected with IVT core, NS3 or green fluorescent protein (GFP) RNA were examined using a DC-specific membrane array. Of the 288 genes on the array, 46 genes were distinctively up- or down-regulated by transfection with IVT core RNA in comparison with NS3 or GFP RNA treatments. Forty-two of these genes are involved in DC maturation. The effects of core on maturation of iDCs were confirmed with a significant increase in surface expression of CD83 and HLA-DR, a reduction of phagocytosis, as well as an increase in proliferation and IFN-γ secretion by T cells in a mixed lymphocyte reaction assay. These results show that HCV core does not have an inhibitory effect on human DC maturation, but could be a target for the immune system.

A Seven-Segmented Influenza A Virus Expressing the Influenza C Virus Glycoprotein HEF

Influenza viruses are classified into three types: A, B, and C. The genomes of A- and B-type influenza viruses consist of eight RNA segments, whereas influenza C viruses only have seven RNAs. Both A and B influenza viruses contain two major surface glycoproteins: the hemagglutinin (HA) and the neuraminidase (NA). Influenza C viruses have only one major surface glycoprotein, HEF (hemagglutinin-esterase fusion). By using reverse genetics, we generated two seven-segmented chimeric influenza viruses. Each possesses six RNA segments from influenza virus A/Puerto Rico/8/34 (PB2, PB1, PA, NP, M, and NS); the seventh RNA segment encodes either the influenza virus C/Johannesburg/1/66 HEF full-length protein or a chimeric protein HEF-Ecto, which consists of the HEF ectodomain and the HA transmembrane and cytoplasmic regions. To facilitate packaging of the heterologous segment, both the HEF and HEF-Ecto coding regions are flanked by HA packaging sequences. When introduced as an eighth segment with the NA packaging sequences, both viruses are able to stably express a green fluorescent protein (GFP) gene, indicating a potential use for these viruses as vaccine vectors to carry foreign antigens. Finally, we show that incorporation of a GFP RNA segment enhances the growth of seven-segmented viruses, indicating that efficient influenza A viral RNA packaging requires the presence of eight RNA segments. These results support a selective mechanism of viral RNA recruitment to the budding site.

BAC transgenic expression efficiency: bicistronic versus ATG‐fusion strategies

Bacterial artificial chromosome (BAC)-based transgene can be expressed bicistronically with the target gene or fused to its translation start codon. To compare the transgene expression efficiencies of these two methods, mice were created that expressed green fluorescent protein (GFP) from the genomic locus of nucleostemin using the bicistronic (NSiGFP) or the ATG-fusion approach (NSmGFP). Three lines with 1, 2, and 4 copies of the NSiGFP transgene, and two lines with 1 copy of the NSmGFP transgene were generated. Of the three NSiGFP lines, only the 4-copy line can match the NSmGFP lines in their GFP protein expression levels. Analyses of the GFP and nucleostemin RNA transcript levels exclude IRES inefficiency and suggest premature termination of the bicistronic message as the cause for low GFP expression in the NSiGFP mice. This work provides important information for designing BAC transgenics when the transgene expression level is crucial.

Adenosine Kinase Inhibition and Suppression of RNA Silencing by Geminivirus AL2 and L2 Proteins

Most plant viruses are initiators and targets of RNA silencing and encode proteins that suppress this adaptive host defense. The DNA-containing geminiviruses are no exception, and the AL2 protein (also known as AC2, C2, and transcriptional activator protein) encoded by members of the genus Begomovirus has been shown to act as a silencing suppressor. Here, a three-component, Agrobacterium-mediated transient assay is used to further examine the silencing suppression activity of AL2 from Tomato golden mosaic virus (TGMV, a begomovirus) and to determine if the related L2 protein of Beet curly top virus (BCTV, genus Curtovirus) also has suppression activity. We show that TGMV AL2, AL2(1-100) (lacking the transcriptional activation domain), and BCTV L2 can all suppress RNA silencing directed against a green fluorescent protein (GFP) reporter gene when silencing is induced by a construct expressing an inverted repeat GFP RNA (dsGFP). We previously found that these viral proteins interact with and inactivate adenosine kinase (ADK), a cellular enzyme important for adenosine salvage and methyl cycle maintenance. Using the GFP-dsGFP system, we demonstrate here that codelivery of a construct expressing an inverted repeat ADK RNA (dsADK), or addition of an ADK inhibitor (the adenosine analogue A-134974), suppresses GFP-directed silencing in a manner similar to the geminivirus proteins. In addition, AL2/L2 suppression phenotypes and nucleic acid binding properties are shown to be different from those of the RNA virus suppressors HC-Pro and p19. These findings provide strong evidence that ADK activity is required to support RNA silencing, and indicate that the geminivirus proteins suppress silencing by a novel mechanism that involves ADK inhibition. Further, since AL2(1-100) is as effective a suppressor as the full-length AL2 protein, activation and silencing suppression appear to be independent activities.

Improved silencing vector co-expressing GFP and small hairpin RNA.

Small interfering RNA (siRNA) is a powerful tool for the specific silencing of gene expression. We developed an improved vector, pG-SUPER, that co-expresses green fluorescent protein (GFP) and small hairpin RNA simultaneously to facilitate analysis of silencing at the level of individual cells. As a test system, we analyzed lamin A/C knockdown in HeLa cells. The GFP signal was a reliable reporter (93%-98%) of strong knockdown (approximately 90%) over a wide range of GFP intensities. The GFP reporter made possible the application of fluorescent-activated cell sorting (FACS) to purify the knockdown cell population. Such populations facilitated Western blotting analysis to determine depletion of the target protein. pG-SUPER was also applied to evaluate gene replacement by exogenous genes rendered refractory to siRNA by introducing silent mutations. Recovery of lamin A was linearly correlated to the expression level of the rescue gene. pG-SUPER will expand plasmid-based siRNA applications through the easy and reliable detection of knockdown and rescued cells.

Functional Genomics in Xenopus laevis: Towards Transgene-Driven RNA Interference and Cell-Specific Transgene Expression

The most direct approach to study the physiological role of a protein of unknown function (Functional Genomics) is to change its expression pattern in an intact organism and analyze the phenotypic consequences of this manipulation. The introduction of a method to generate stably transgenic Xenopus laevis has paved the way to the use of tissue / cell- and developmental stage-specific promoters allowing to study the physiological function of proteins in a defined set of fully differentiated cells. Whereas stable (over)expression of proteins in Xenopus is now within reach, stable inhibition of protein expression can only be accomplished randomly, by gene trap approaches. We here report our efforts to induce stable RNA interference (RNAi) in X. laevis via transgene-driven expression of inverted repeats. Stable, and muscle- and neuron-specific knock-down of expression of exogenous green fluorescent protein (GFP) reporter was achieved via RNA polymerase II promoter-driven expression of long GFP RNA duplexes. Unfortunately, our attempts to induce RNAi directed against various endogenous targets, based on the use of RNA polymerase II and III promoters, and long and short inverted repeats have not resulted in a reliable protocol for stable, transgene-driven RNAi in Xenopus. In the second part, we present an example of the use of a cell-specific promoter for functional studies. Cell-specific transgene overexpression of a GFP-tagged member of the p24 family thought to be involved in intracellular protein transport was achieved and this manipulation of the intermediate pituitary melanotrope cell had a phenotypic consequence at its physiological target, the skin melanophore. Thus, the traditional experimental advantages of X. laevis combined with the recently developed technique of stable, non-mosaic Xenopus transgenesis make this lower vertebrate an attractive model organism for Functional Genomics. Keywords: dsrna, intermediate pituitary cells, p24

Spreading of RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase.

RNA silencing is a sequence-specific RNA degradation process that follows the recognition of double-stranded RNA. Here, we show that virus vectors carrying parts of a green fluorescent protein (GFP) transgene targeted RNA silencing in Nicotiana benthamiana and Arabidopsis against the entire GFP RNA. These results indicate that there was spreading of RNA targeting from the initiator region into the adjacent 5' and 3' regions of the target gene. Spreading was accompanied by methylation of the corresponding GFP DNA. It also was dependent on transcription of the transgene and on the putative RNA-dependent RNA polymerase, SDE1/SGS2. These findings indicate that SDE1/SGS2 produces double-stranded RNA using the target RNA as a template. RNA silencing of ribulose-1,5-bisphosphate carboxylase/oxygenase and phytoene desaturase was not associated with the spreading of RNA targeting or DNA methylation, indicating that these endogenous RNAs are not templates for SDE1/SGS2.

Identifying proteins that affect mRNA localization in living cells

Messenger RNA transport has emerged as a significant mechanism for regulating gene expression. Many of the protein factors affecting RNA transport remain unknown. The emergence of green fluorescent protein (GFP) fluorescence microscopy allows imaging in living cells and an increased understanding of in vivo molecular transport. GFP imaging is now applied to RNA transport by engineering RNA hairpins into the RNA of interest and observing fluorescence from GFP fused to an RNA-binding protein that recognizes the hairpins. In yeast, different genetic backgrounds can be tested to identify various proteins that affect RNA transport and localization. The technology also allows the swapping of different regions of the RNA to determine the cis requirements for transport. GFP RNA imaging opens many possibilities to examine RNA transport in real time in a variety of different organisms.

Overexpression of agrin isoforms in Xenopus embryos alters the distribution of synaptic acetylcholine receptors during development of the neuromuscular junction.

Synapse formation involves a large number of macromolecules found in both presynaptic nerve terminals and postsynaptic cells. Many of the molecules involved in synaptogenesis of the neuromuscular junction have been discovered through morphological localization to the synapse and functional cell culture assays, but their role in embryonic development has been more difficult to study. One of the best understood of these molecules is agrin, a synaptic extracellular matrix protein secreted by both motor neurons and muscle cells, that organizes the postsynaptic apparatus, including high-density aggregates of acetylcholine receptors (AChRs), at the neuromuscular junction. We tested the specific hypothesis that different agrin isoforms made by neurons and muscle cells contribute to agrin's synapse organizing activity in the embryo. Agrin isoforms were overexpressed by injecting synthetic RNA into Xenopus laevis embryos at the one- or two-cell stage. To mark cells containing agrin RNA, green fluorescent protein (GFP) RNA was coinjected. The relative area of muscle AChR aggregates was measured by confocal microscopy and image analysis in GFP-positive segments of injected embryos. Innervated regions of myotomal muscles were compared in animals injected with a mixture of agrin and GFP RNAs or with GFP RNA alone. Overexpression of COOH-terminal 95-kDa fragments of a rat agrin isoform made only by neurons (4,8) and the major isoform (0,0) made by muscle cells both increased AChR cluster area by 100-200%. Rat agrin protein was colocalized with AChR aggregates in innervated regions of muscles in injected embryos. These results show that agrin derived from both the nerve terminal and the muscle cell could contribute to synaptic differentiation at the embryonic neuromuscular junction. They further demonstrate the usefulness of overexpression by RNA injection as an assay for molecular function in embryonic synapse formation.