In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells

Summary

We present a protocol to study mRNA translation regulation in poxvirus-infected cells using in vitro Transcribed RNA-based luciferase reporter assay. The assay can be used for studying translation regulation by cis-elements of an mRNA, including 5’-untranslated region (UTR) and 3’-UTR. Different translation initiation modes can also be examined using this method.

Abstract

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5'-poly(A) leader in the 5'-UTR. 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 of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the 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 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 investigated using this method.

Introduction

According to the central dogma, genetic information flows from DNA to RNA and then finally to protein^1,2. This flow of genetic information is highly regulated at many levels including mRNA translation^3,4. Development of reporter assays to measure regulation of gene expression will facilitate understanding of regulatory mechanisms involved in this process. Here we describe a protocol to study mRNA translation using an in vitro transcribed 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 synthesis^6,7,8. To efficiently synthesize viral proteins, viruses evolved many strategies to hijack cellular translational machinery to redirect it for translation of viral mRNAs^7,8. One commonly employed mechanism by viruses is to use cis-acting elements in their transcripts. Notable examples include the Internal Ribosome Entry Site (IRES) and cap-independent translation enhancer (CITE)^9,10,11. These cis-elements render the viral transcripts a translational advantage by attracting translational machinery via diverse mechanisms^12,13,14. Over 100 poxvirus mRNAs have an evolutionarily conserved cis-acting element in the 5’-untranslated region (5’-UTR): a 5’-poly(A) leader at the very 5’ ends of these mRNAs^15,16. The lengths of these 5’-poly(A) leaders are heterogeneous and are generated by slippage of the poxvirus-encoded RNA polymerase during transcription^17,18. 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 poxviruses^19,20.

The in vitro transcribed RNA-based luciferase reporter assay was initially developed to understand the role of 5’-poly(A) leader in mRNA translation during poxvirus infection^19,21. Although plasmid DNA-based luciferase reporter assays have been widely used, there are several drawbacks that will complicate the result interpretation in poxvirus-infected cells. First, plasmids are able to replicate in VACV-infected cells²². Second, cryptic transcription often occurs from plasmid DNA^18,23,24. Third, 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 in vitro transcribed RNA-based luciferase reporter assay circumvents these issues and the data interpretation is straightforward.

There are four key steps in this method: (1) polymerase chain reaction (PCR) to generate the DNA template for in vitro transcription; (2) in vitro transcription 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 in vitro transcription using T7 polymerase. During in vitro transcription, m⁷G cap or other cap analog is incorporated in newly synthesized mRNA. The capped transcripts are transfected into uninfected or VACV-infected cells. The cell lysate is collected at the desired time after transfection to measure luciferase activities that indicate 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, the in vitro transcribed 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

1. Preparation of DNA Template by PCR for In Vitro Transcription

1. To prepare the DNA template by PCR, design primers. When designing primers consider crucial characteristics like primer length, annealing temperature (T_m), GC content, 3’ end with G or C etc.
   NOTE: Discussed in detail in these literature^25,26,27.
2. Design primers to generate PCR amplicon containing the following elements in 5’ to 3’ direction: T7-Promoter, poly(A) leader, firefly luciferase ORF and a poly(A) tail referred hereafter to as T7_12A-Fluc. Design primers (Forward and Reverse) to encompass all the additional elements not present in the template DNA (Figure 2A).
   NOTE: The sequence of all elements can be found in Table 1.
3. Include several extra nucleotides in forward primer (5’-3’)²⁸, followed by T7-promoter, poly(A) leader or desired 5’-UTR sequence and approximately 20 nucleotides, adjust based on T_m, corresponding to the 5’ end of the reporter gene’s ORF. Ensure 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 overlap extension PCR²⁹.
4. Design reverse primer (5’-3’) to include poly(A) tail and approximately 20 nucleotides, adjust based on T_m, corresponding to the 3’ end of the reporter gene’s ORF. Ensure the corresponding region in the primer is identical to the anti-sense strand (- strand) of the gene and an in-frame stop codon is present before the poly(A) tail.
   NOTE: The desired length of A’s in a 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.
5. For internal control, design another set of primers containing the following elements in 5’ to 3’ direction: T7 Promoter, a random 5’-UTR coding sequence containing Kozak sequence, Renilla luciferase ORF and poly(A) tail referred hereafter to as T7_Kozak-Rluc.
6. In a PCR tube, add the reagents in the following order: DNase free water, 2x high-fidelity DNA polymerase, primers, and sequence confirmed luciferase template DNA (Table 2).
   NOTE: Amounts of individual components in the mixture should be adjusted according to the reaction volume.
7. Use a standard 3-step (Denaturation, Annealing, Extension) PCR cycle to generate a DNA template as shown in Table 3.
   NOTE: Annealing temperature X °C depends on the primer set being used and extension time T min depend on the PCR amplicon size and DNA polymerase used.
8. Detect the PCR product by running 5-10% of PCR reaction in 1% agarose Tris-acetate-EDTA (TAE) gel electrophoresis (containing 0.1µg/ml ethidium bromide) along with commercially available molecular weight standard. Visualize the gel under a UV illuminator to determine the size of the PCR product.
9. After determining the correct size of the PCR product, ~1.7 kb for T7_12A-Fluc and ~1.0 kb for T7_Kozak-Rluc, purify it using a commercially available PCR purification kit. Elute the DNA using 100 µL nuclease free water (Figure 2B).
10. Once purified, check the concentration of the DNA using a spectrophotometer and determine the A260/A280 ratio (~1.8-2.0 is acceptable).
11. Store purified DNA at -20 °C or use for in vitro transcription immediately.

2. Generate mRNA by In Vitro Transcription

1. Synthesize RNA from the PCR product in vitro, using an in vitro transcription kit (Figure 3A).
   NOTE: T7_12A-Fluc and T7_Kozak-Rluc DNA templates are used to synthesize 12A-Fluc and Kozak-Rluc mRNAs, respectively.
   1. To do this, take a microcentrifuge tube and add the reagents in the following order: DNase-RNase free water, NTP Buffer Mix, Cap Analog, Template PCR Product, T7-RNA polymerase Mix (Table 4).
      NOTE: Other capping systems can also be used to cap RNA sequentially after in vitro transcription following the manufacturer’s instruction.
   2. Mix thoroughly and incubate at 37 °C for 2 h.
   3. Proceed to the purification of the synthesized RNA using an RNA purification kit.
2. Run purified RNA in 1.5% agarose Tris-borate-EDTA (TBE) gel (containing 0.5 µg/mL ethidium bromide) to check the RNA. Visualize the gel under a UV illuminator (Figure 3B).
3. Check the concentration of the RNA using a spectrophotometer and determine the A260/A280 ratio (~1.8-2.0 is acceptable).
4. Aliquot the purified RNA and store at -80 °C.

3. Transfect mRNA to Cells

1. Seed HeLa cells in a 24-well plate (to be approx. ~80-90% confluent next day) and incubate overnight in an incubator at 37 °C with 5% CO₂.
2. Infect HeLa cells with vaccinia virus (VACV) at a Multiplicity of Infection (MOI) of 5 or keep uninfected HeLa cells for comparison.
   NOTE: MOI is the number of infectious viral particles per cell.
3. MOI of X= {[(Number of cells * X) / Virus Titer] * 1000} µl of virus per 1 mL medium.
4. After desired h post infection (hpi) (in this experiment at 10-12 hpi), transfect mRNA (500 ng of total mRNA per well of 24-well plates) using a cationic lipid transfection reagent as shown in Figure 3C.
   1. For one well of a 24-well plate, mix 480 ng of 12A sequence bearing firefly luciferase (12A-Fluc) mRNA and 20 ng of Kozak sequence bearing Renilla luciferase (Kozak-Rluc) mRNA in one microcentrifuge tube. In another microcentrifuge tube add 1.1 µL of cationic lipid transfection reagent.
   2. Add 55 µL of reduced serum medium in both tubes. Mix and incubate at room temperature for 5 min.
   3. After 5 min of incubation, add 55 µL cationic lipid transfection reagent containing reduced serum medium in mRNA containing tube.
   4. Mix gently but thoroughly, and incubate at room temperature for 15 min.
   5. During the incubation, remove the cell culture medium and add 400 µL of reduced serum medium per well of 24-well plates.
   6. After incubation, add 100 µL of the mixture dropwise and evenly to one well of 24-well plates.

4. Measure Luciferase Activities

1. Five-hours post-co-transfection of 12A-Fluc and Kozak-Rluc mRNA, measure luciferase activity using a luciferase assay system capable of performing two reporter assays (e.g., Dual Luciferase Reporter assay kit).
2. Remove the reduced serum medium and lyse the cells by adding 150 µL 1x lysis buffer, a component of the luciferase assay kit.
3. After 10 min incubation at room temperature, collect the lysate by scrapping the cells and transfer to a microcentrifuge tube.
4. Centrifuge the lysate at 12,000 x g for 10 min at 4 °C to pellet cell debris.
5. Add 30 µL of supernatant in opaque-walled 96 well white assay plate with a solid bottom.
6. Measure the dual luminescence using the luciferase assay kit and a multimode plate reader luminometer.
7. Perform the measurement using kinetics function (on a per-well basis) using the settings described in Table 5.
   NOTE: The reading can also be taken using manual luminometer. Add an equal volume of lysate and substrate for Fluc in a cuvette. Wait for 2 s and measure for 10 s using luminometer. Following Fluc measurement, quickly take out cuvette from luminometer and add an equal volume of the substrate for Rluc manually. Again, wait for 2 s and measure for 10 s using luminometer.
8. Export the luminescence reading data into a desirable file format.
9. Determine relative translation rate from 12A-Fluc mRNA in uninfected and VACV infected HeLa cells by dividing Fluc value by internal control Rluc value.
   NOTE: Supplementary Figure 1 shows the step-by-step analysis of raw data to get relative Fluc activity.

Results

The four steps of in vitro transcribed RNA-based luciferase reporter assay: PCR to generate DNA template for In vitro transcription, in vitro transcription 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...

Discussion

All four-core steps are critical to the success of the in vitro transcribed 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...

Materials

Name	Company	Catalog Number	Comments
2X-Q5 Master mix	New England Biolabs	M0492	High-Fidelity DNA Polymerase used in PCR
3´-O-Me-m7G(5')ppp(5')G RNA Cap Structure Analog	New England Biolabs	S1411L	Anti reverse Cap analog or ARCA
Corning 96 Well Half-Area white flat bottom polystyrene microplate	Corning	3693	Opaque walled 96 well white plate with solid bottom
Dual-Luciferase Reporter Assay System	Promega	E1960	Dual-Luciferase Assay Kit (DLAK)
E.Z.N.A. Cycle Pure Kit	OMEGA BIO-TEK	D6492	PCR purification kit
GloMax Navigator Microplate Luminometer	Promega	GM2010	Referred as multimode plate reader luminometer
HiScribe T7 Quick High Yield RNA synthesis Kit	New England Biolabs	E2050S	In-Vitro transcription kit
Lipofectamine 2000	Thermo Fisher Scientific	11668019	Cationic lipid transfection reagent
NanoDrop2000	Thermo Fisher Scientific	ND-2000	Used to measure DNA and RNA concentration
Opti-MEM	Thermo Fisher Scientific	31985070	Reduced serum media
Purelink RNA Mini Kit	Thermo Fisher Scientific	12183018A	RNA purification kit
Vaccinia Capping System	New England Biolabs	M2080	Capping system