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....

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 h....

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