TITLE:In-Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-Infected Cells  KEYWORDS:In-Vitro Transcription, Luciferase assay, Vaccinia virus, Poxvirus, Translation, 5’-UTR, 5’-poly(A) leaderSUMMARY:In-Vitro Transcribed (IVT) RNA-based luciferase reporter assay enables studying the regulation of mRNA translation in poxvirus-infected cells. The assay can be used to study the translation regulated by cis-elements present in an mRNA, including 5’-untranslated region (UTR) and/or 3’-UTR.ABSTRACT:Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5’-poly(A) leader in the 5’-UTR. In this study, to dissect the role of 5’-poly(A) leader in mRNA translation during poxvirus infection, we developed an in-vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises four core steps: (1) PCR to amplify the DNA template for in-vitro transcription; (2) In-vitro transcription to generate capped mRNA using T7 RNA polymerase and cap analog; (3) Transfection to deliver in-vitro transcribed mRNA into cells; (4) Detection of luciferase activity as indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5’-UTR and/or 3’-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be examined using this method.INTRODUCTION:According to the central dogma, genetic information flows from DNA to RNA and then finally to protein1,2. This flow of genetic information is highly regulated at many levels including mRNA translation3,4. Development of reporter assays to quickly measure regulation of gene expression will facilitate understanding of the regulatory mechanisms involved in this process. Here we describe a protocol to study mRNA translation using an in-vitro transcribed (IVT) RNA-based luciferase reporter assay in poxvirus-infected cells.  Poxviruses comprise many highly dangerous human and animal pathogens5. Like all other viruses, poxviruses exclusively rely on host cell machinery for protein synthesis6–8. To efficiently synthesize viral proteins, viruses evolved many strategies to hijack cellular translational machinery to redirect them for translation of viral mRNAs7,8. One commonly employed mechanism by viruses is to use cis-acting elements in their transcripts. Notable examples include Internal Ribosome Entry Site (IRES), cap?independent translation enhancer (CITE), etc.9–11. These cis-elements render the viral transcripts a translational advantage by attracting translational machinery via diverse mechanisms12–14. Over 100 poxvirus mRNAs have an evolutionarily conserved cis-acting element in the 5’-untranslated regions (5’-UTRs): a 5’-poly(A) leader at the very 5’ ends of these mRNAs15,16. The lengths of these 5’-poly(A) leaders are heterogeneous and are generated by slippage of the poxvirus-encoded RNA polymerase during transcription17,18. We, and others, recently discovered that the 5’-poly(A) leader confers a translation advantage to an mRNA in cells infected with vaccinia virus (VACV), the prototypic member of poxviruses19,20.   The IVT RNA-based luciferase reporter assay was initially developed to understand the role of 5’-poly(A) leader in mRNA translation during poxvirus infection19,21. Although plasmid DNA-based luciferase reporter assays have been widely used, there are several drawbacks that will complicate the result interpretation. First, plasmids are able to replicate in VACV-infected cells22. Secondly, cryptic transcription often occurs from plasmid DNA18,23,24. Thirdly, VACV promoter-driven transcription generates poly(A)-leader of heterogeneous lengths consequently making it difficult to control the poly(A)-leader length in some experiments18. An IVT RNA-based luciferase reporter assay will circumvent these issues and the data interpretation is straightforward.  There are four key steps in this methods: (1) polymerase chain reaction (PCR) to generate the DNA template for in-vitro transcription (IVTn); (2) IVTn to generate mRNA; (3) transfection to deliver mRNA into cells; and (4) Detection of luciferase activity as indicator of translation (Figure 1). The resulting PCR amplicon contains the following elements in 5’ to 3’ direction: T7-Promoter, poly(A) leader or desired 5’-UTR sequence, firefly luciferase open reading frame (ORF) followed by a poly(A) tail.  PCR amplicon is used as the template to synthesize mRNA by IVTn using T7 polymerase. During IVTn, m7G cap or other cap analog is incorporated in newly synthesized mRNA. The capped transcripts are transfected into uninfected or VACV-infected cells. Cell lysate is collected at desired time after transfection to measure luciferase activities indicating protein production from transfected mRNA. This reporter assay can be used to study translation regulation by cis-element present in 5’-UTR, 3’-UTR or other regions of an mRNA. Furthermore, IVT RNA-based assay can be used to study different mechanisms of translation initiation including cap-dependent initiation, cap-independent initiation, re-initiation and internal initiation like IRES. PROTOCOL:Note: Information about the Material/Equipment used in this protocol can be found in Table of Materials.Prepare DNA template by PCR for in vitro transcriptionConsider crucial characteristics when designing primers discussed in these literature25–27.Design primers to generate PCR amplicon containing following elements in 5’ to 3’ direction: extra nucleotides, T7-Promoter, poly(A) leader, firefly luciferase ORF and a poly(A) tail. Primers (Forward and Reverse) need to encompass all the additional elements not present in the template DNA (Figure 2A).Design another set of primers for internal control of transfection efficiency containing the following elements in 5’ to 3’ direction: extra nucleotides, T7 Promoter, a random 5’-UTR coding sequence containing Kozak sequence, renilla luciferase ORF and poly(A) tail.Design forward primer (5’-3’) that includes several extra nucleotides28, T7-promoter, poly(A) leader or desired 5’-UTR sequence and approximately 20 nucleotides corresponding to the 5’ end of the reporter gene’s ORF. Make sure the corresponding region in the primer is identical to the sense strand (+ strand) of the gene.Note:For long 5’-UTR, synthesize two DNA fragments: one with T7 promoter followed by long 5’-UTR and second with reporter gene’s ORF. Join these two fragments using overlapping PCR29.1.5.  Design reverse primer (5’-3’) that includes a poly(A) tail and approximately 20 nucleotides corresponding to the 3’ end of the reporter gene’s ORF. Make sure the corresponding region in the primer is identical to the anti-sense strand (- strand) of the gene and a stop codon is present before the poly(A) tail.Note:The desired length of A’s in poly(A) leader or poly(A) tail can be customized in the primers. For example, to add 50 A’s in the poly(A) tail, the reverse primer should entail 50 T’s. Similarly, to add 20 A’s in the poly(A) leader, the forward primer should entail 20 A’s.The length of the nucleotide corresponding to the reporter gene’s ORF (5’ or 3’ end) should be adjusted based on the annealing temperature (Tm).The sequence of all elements can be found in Table 1.1.6.  In a PCR tube, add the reagents in the following order:1.6.1.      DNase free water:      38 µl1.6.2.      2X-Q5 Master mix:     50  µl1.6.3.      Forward Primer (10 µM):     4 µl1.6.4.      Reverse Primer (10 µM):     4 µl1.6.5.      Luciferase DNA Template (1-10 ng/µl):      4 µl 1.6.6.      Total:       100 µlNote:Other High-Fidelity DNA polymerase product will suffice and the amounts of individual components in the mixture should be adjusted accordingly.DNA template should be confirmed with correct sequence.1.7.  Use a standard 3-step (Denaturation, Annealing, Extension) PCR cycle to generate desired PCR amplicon:1.7.1.      Initial denaturation 95 °C:    2 minutes   (1X Cycle)1.7.2.      Denaturation  95 °C:    15 seconds 1.7.3.      Annealing   X °C:    30 seconds   (25X Cycle)1.7.4.      Extension   72 °C:     T minutes  1.7.5.      Final Extension  72 °C:     7 minutes   (1X Cycle)1.7.6.      Hold   4 °C:     ?Note:Annealing temperature X °C depends on the primer set being used.Extension time T minutes depend on the PCR amplicon size and DNA polymerase used.1.8.  Detect the PCR product by running aliquots (10%) of PCR reaction in 1% agarose gel electrophoresis (containing 0.1 µg/ml ethidium bromide) along with commercially available molecular weight standard. Visualize the gel under UV illuminator to determine the size of the PCR product.1.9.  After determining the correct size of the PCR product, purify it by using a commercially available PCR purification kit.1.10.                    Once purified, determine the concentration of DNA and A260/A280 ratio (Ideally 2.0, ~1.8 is acceptable).1.11.                    Purified DNA can be stored at -20 °C or used for IVTn immediately.Generate mRNA by IVTnSynthesize RNA from PCR product in vitro, using an in-vitro transcription kit.Note:HiScribe T7 Quick High Yield RNA Synthesis Kit is used in this protocol. Other in-vitro transcription kits should work as well.2.2.  In a microcentrifuge tube, add the reagents in the following order:2.2.1.      DNase-RNase free water:    X µl2.2.2.      NTP Buffer Mix (20 mM of each NTP):  2 µl 2.2.3.      Cap Analog (Stock 40 mM):    4 µl2.2.4.      Template PCR Product (400 ng):   X µl 2.2.5.      T7-RNA polymerase Mix:               2 µl2.2.6.      Total:       20 µlNote:Volume X µl depends on the concentration of the Template PCR Product.Vaccinia Capping System can also be used to cap RNA sequentially after IVTn.2.3.  Mix thoroughly and incubate at 37 °C for 2 hours.2.4.  Proceed with the purification of the synthesized RNA using an RNA purification kit.2.5.  Determine the concentration of RNA and A260/A280 ratio (Ideally ~2).2.6.  The purified RNA can be stored at -80 °C.Transfect mRNA to cells Seed cells in 24 well plate (To be approx. ~80-90% confluent next day) and incubate overnight in the incubator at 37 °C and 5% CO2.Infect cells with VACV at a Multiplicity of Infection (MOI) of 5.After desired hours post infection (hpi) (we often transfect at 10-12 hpi).  Transfect mRNA (500 ng of total mRNA per well of 24 well plates) using cationic lipid transfection reagent as shown in Figure 3.   For one well of 24 well plates, mix 480 ng of firefly luciferase (Fluc) mRNA and 20 ng of renilla luciferase (Rluc) mRNA in one microcentrifuge tube. In another microcentrifuge tube add 1.1 µl of cationic lipid transfection reagent.   Add 55 µl of reduced serum media in both tubes. Mix and incubate in room temperature for 5 minutes.   After 5 minutes of incubation, add 55 µl cationic lipid transfection reagent containing reduced serum media in mRNA containing tube.   Mix thoroughly and incubate in room temperature for 15 minutes.   During the incubation, remove the cell culture media and add 400 µl of reduced serum media per well of 24 well plates.   After incubation, add 100 µl of the mix to one well of 24 well plates.Measure luciferase activities4.1. Five-hour post-co-transfection of Fluc and Rluc mRNA, measure luciferase activity using          a Dual-Luciferase Reporter Assay System (DLAS).4.2  Remove reduced serum media and lyse the cells by adding 150 µl 1X Passive lysis buffer, a component of DLAS.4.3.      After 10 minutes incubation at room temperature, collect the lysate by scrapping the cells and transfer to microcentrifuge tube.4.4.      Centrifuge the lysate at 12,000 X g for 10 minutes at 4 °C to pellet cell debris.4.5.      Add 30 µl of supernatant in opaque-walled 96 well white assay plate with a solid bottom.4.6.      Measure the dual luminescence using DLAS in multimode plate reader luminometer.4.7.      The measurement is taken using kinetics function (all steps on a per-well basis) using the following settings:Inject Luciferase Assay Substrate (Fluc):  30 µlWait / Incubation time:    2 secLuminescence Measurement (Fluc):   10 secondsStop & Glo Substrate (Rluc):    30 µlWait / Incubation time:    2 secLuminescence Measurement (Rluc):   10 seconds4.8.      Export the luminescence reading data into an excel file. 4.9.      Analyze the data.REPRESENTATIVE RESULTS:The four steps of IVT RNA-based luciferase reporter assay:  PCR to generate DNA template for IVTn, IVTn to generate mRNA, mRNA transfection, and luciferase measurement, can be seen in the schematic diagram (Figure 1). Designing of primers for both DNA templates (Fluc and Rluc) and the general scheme of overhang extension PCR is illustrated in the schematic (Figure 2A). After PCR, the correct sized PCR product is detected by agarose gel electrophoresis (Figure 2B). Subsequently, the PCR product is used as the template to synthesize RNA in-vitro (Figure 3A), which is then purified and transfected into cells using cationic lipid transfection reagent (Figure 3B).The IVT RNA-based luciferase reporter assay was developed to understand the role of 5’-poly(A) leader in mRNA translation during poxvirus infection. Using this assay, we tested the translation efficiency of a Fluc mRNA that contains a 5’-poly(A) leader (12nt)  in uninfected and VACV-infected cells. The Fluc value was normalized using Rluc value in both uninfected and VACV infected cells to determine the relative Fluc activity (i.e. Fluc activity/Rluc activity) (Figure 4A). The division of Fluc by Rluc normalized the transfection efficiency and RNA stability in a particular well. Using this analysis approach, we determined that 5’-poly(A) leader containing mRNA has a translational advantage during VACV infection (Figure 4B). The advantage in infected cells was not due to differential transfection efficiency or mRNA stability as the RNA level was similar in uninfected and VACV infected cells 5 hours post mRNA transfection19.FIGURE AND TABLE LEGENDS:Figure 1: Schematic of the experimental procedure.  PCR is used to generate a DNA template with desired elements. mRNA encoding a luciferase reporter gene is synthesized in-vitro using a T7-RNA polymerase based system. A Firefly luciferase (Fluc) mRNA is co-transfected with a Renilla luciferase (Rluc) mRNA into uninfected or VACV-infected cells. Luciferase activities are measured using a luminometer with dual luciferase capability.Figure 2: Primer design and PCR-based DNA amplification.  (A) Forward primer is synthesized to include 8nt upstream of the T7 promoter followed by a desired 5’-UTR and part of the 5’ end of the luciferase reporter gene, while the reverse primer includes a poly(A) tail and the 3’ end of the luciferase reporter gene. By overhang extension PCR using a plasmid template containing luciferase gene, a DNA template is generated. (B) DNA band of the desired size from PCR reaction was detected using 1% agarose gel electrophoresis.Figure 3: mRNA synthesis and transfection.  (A) Schematic of in-vitro transcription. DNA amplified by PCR containing the luciferase gene downstream from the 5’-UTR of interest and the T7 promoter is used as a template.  The T7 RNA polymerase is recruited to the promoter and adds ribonucleotides, shown in white, from 5’ to 3’ direction. Once mRNA is 25-30nt long m7G cap is added using an anti-reverse cap analog, ARCA. (B) Schematic demonstrating the transfection of reporter mRNA into cells. Medium containing either the reporter mRNA or cationic lipid transfection reagent in separate tubes is allowed to equilibrate at room temperature for 5 minutes. The solutions are then mixed followed by incubation at room temperature for 15 minutes after which the RNA/transfection reagent mixture is added into cells in culture plates.Figure 4: Increased translational efficiency of mRNA containing a 5’-poly(A) leader.  (A) Fluc mRNA containing a poly(A) leader in the 5’-UTR and Rluc mRNA with the Kozak consensus sequence in the 5’-UTR are co-transfected into cells. (B) Fluc mRNA with 5’-poly(A) leader was transfected in uninfected and VACV infected cells. Five-hours post-co-transfection, luciferase activity was measured using a luminometer.Table 1. The sequence of different elements. The table contains the sequence of T7-Promoter, poly(A) leader, Kozak sequence, firefly luciferase ORF, renilla luciferase ORF, poly(A) tail element in reporter PCR amplicon.DISCUSSION:All four core steps: PCR, IVTn, mRNA transfection and luciferase assay, are critical to the success of IVT RNA-based luciferase reporter assay. Special attention should be given to primer design, especially for the T7 promoter sequence. T7 RNA Polymerase starts transcription from the underlined first G (GGG-5’UTR-AUG-) in T7 promoter added before the 5’-UTR sequence. Although the transcription start site (TSS) starts from the first G at the 5’ end, decreasing the number of G’s less than three in T7 promoter region decreased the RNA yield/output from IVTn. During the experiment, we observed that gel purified DNA product was not the best for IVTn as both yield and quality of RNA are lower. To work around this complication, we only ran 5-10% of the PCR reaction in 1% agarose gel electrophoresis to determine the size and purified the rest 90-95%, using a PCR purification kit, to be used for IVTn. The proposed method is suitable for use in different model systems with some modifications like the method of mRNA delivery, internal control to be used, a suitable time for translation, sample preparation and analysis of data. Currently, this main limitation of this method is that it is an in-vitro assay to quickly test translation regulation by cis elements. We would like to emphasize this method should be corroborated by other in-vivo experiments, if possible. Compared to DNA modification, the roles of RNA modifications are less studied. However, with the discovery of enzymes that write, read and erase RNA modifications30–35, it is now possible to study the influence of RNA modification in gene expression. The IVT RNA-based luciferase reporter assay can be modified to incorporate different RNA modifications and used to test their effect on RNA translation. First, this method can incorporate different cap analogs that have various modifications30. Additionally, supplementing an internal RNA modifying enzyme during or after IVTn can incorporate internal RNA modification. Addition of a modification to cap 0, cap 1, and an internal RNA modification will provide a tool to study the role of these RNA modifications in translation.The IVT RNA-based luciferase reporter assay has great potential and broad applications in understanding basic biology about RNA translation. Different mechanisms for the initiation of translation, including cap-dependent initiation, cap-independent initiation, re-initiation and internal initiation such as IRES can be studied using this method. 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