The project was funded by the National Institutes of Health (AI128406 www.nih.gov) to ZY and in part by Johnson Cancer Research Center (http://cancer.k-state.edu) in the form of Graduate Student Summer Stipend to PD.

1. Preparation of DNA Template by PCR for In Vitro Transcription
<ol>
	<li>To prepare the DNA template by PCR, design primers. When designing primers consider crucial characteristics like primer length, annealing temperature (Tm), GC content, 3&#8217; end with G or C etc. 
	NOTE: Discussed in detail in these literature25,26,27.</li>
	<li>Design primers to generate PCR amplicon ...

<ol>
	<li>Crick, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+protein+synthesis.">On protein synthesis.</a> Symposia of the Society for Experimental Biology. 12, 138-163 (1958).</li><li>Crick, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+Dogma+of+Molecular+Biology.">Central Dogma of Molecular Biology.</a> Nature. 227, 561-563 (1970).</li><li>Sonenberg, N., Hinnebusch, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Translation+Initiation+in+Eukaryotes:+Mechanisms+and+Biological+Targets.">Regulation of Translation Initiation in Eukaryotes: Mechanisms and Biological Targets.</a> Cell. 136, 731-745 (2009).</li><li>Spriggs, K. A., Bushell, M., Willis, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+Regulation+of+Gene+Expression+during+Conditions+of+Cell+Stress.">Translational Regulation of Gene Expression during Conditions of Cell Stress.</a> Molecular Cell. 40, 228-237 (2010).</li><li>Shchelkunov, S. N., Marennikova, S. S., Moyer, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Orthopoxviruses Pathogenic for Humans. , (2005).</li><li>Gale, M., Tan, S. L., Katze, M. 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B., Mohr, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tinkering+with+Translation:+Protein+Synthesis+in+Virus-Infected+Cells.">Tinkering with Translation: Protein Synthesis in Virus-Infected Cells.</a> Cold Spring Harbor Perspectives in Biology. 5, a012351 (2013).</li><li>Cao, S., Dhungel, P., Yang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Going+against+the+Tide:+Selective+Cellular+Protein+Synthesis+during+Virally+Induced+Host+Shutoff.">Going against the Tide: Selective Cellular Protein Synthesis during Virally Induced Host Shutoff.</a> Journal of Virology. 91, e00071-e00017 (2017).</li><li>Pelletier, J., Kaplan, G., Racaniello, V. R., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cap-independent+translation+of+poliovirus+mRNA+is+conferred+by+sequence+elements+within+the+5&#8217;+noncoding+region.">Cap-independent translation of poliovirus mRNA is conferred by sequence elements within the 5&#8217; noncoding region.</a> Molecular and Cellular Biology. 8, 1103-1112 (1988).</li><li>Pelletier, J., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Internal+initiation+of+translation+of+eukaryotic+mRNA+directed+by+a+sequence+derived+from+poliovirus+RNA.">Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA.</a> Nature. 334, 320-325 (1988).</li><li>Guo, L., Allen, E., Miller, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+a+cap-independent+translation+element+that+functions+in+either+the+3&#8242;+or+the+5&#8242;+untranslated+region.">Structure and function of a cap-independent translation element that functions in either the 3&#8242; or the 5&#8242; untranslated region.</a> RNA. 6, 1808-1820 (2000).</li><li>Simon, A. E., Miller, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3&#8242;+Cap-Independent+Translation+Enhancers+of+Plant+Viruses.">3&#8242; Cap-Independent Translation Enhancers of Plant Viruses.</a> Annual Review of Microbiology. 67, 21-42 (2013).</li><li>Marom, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+poly(A)+binding+proteins+mediate+internal+translational+initiation+by+a+plant+viral+IRES.">Diverse poly(A) binding proteins mediate internal translational initiation by a plant viral IRES.</a> RNA Biology. 6, 446-454 (2009).</li><li>Liu, B., Qian, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+reprogramming+in+cellular+stress+response.">Translational reprogramming in cellular stress response.</a> Wiley Interdisciplinary Review. RNA. 5, 301-305 (2014).</li><li>Ahn, B. Y., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capped+poly(A)+leaders+of+variable+lengths+at+the+5&#8217;+ends+of+vaccinia+virus+late+mRNAs.">Capped poly(A) leaders of variable lengths at the 5&#8217; ends of vaccinia virus late mRNAs.</a> Journal of Virology. 63, 226-232 (1989).</li><li>Ahn, B. Y., Jones, E. V., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+vaccinia+virus+gene+encoding+an+18-kilodalton+subunit+of+RNA+polymerase+and+demonstration+of+a+5&#8217;+poly(A)+leader+on+its+early+transcript.">Identification of the vaccinia virus gene encoding an 18-kilodalton subunit of RNA polymerase and demonstration of a 5&#8217; poly(A) leader on its early transcript.</a> Journal of Virology. 64, 3019-3024 (1990).</li><li>Schwer, B., Visca, P., Vos, J. C., Stunnenberg, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discontinuous+transcription+or+RNA+processing+of+vaccinia+virus+late+messengers+results+in+a+5&#8242;+poly(A)+leader.">Discontinuous transcription or RNA processing of vaccinia virus late messengers results in a 5&#8242; poly(A) leader.</a> Cell. 50, 163-169 (1987).</li><li>Yang, Z., Martens, C. A., Bruno, D. P., Porcella, S. F., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pervasive+initiation+and+3&#8242;+end+formation+of+poxvirus+post-replicative+RNAs.">Pervasive initiation and 3&#8242; end formation of poxvirus post-replicative RNAs.</a> Journal of Biological Chemistry. 287, 31050-31060 (2012).</li><li>Dhungel, P., Cao, S., Yang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+5&#8217;-poly(A)+leader+of+poxvirus+mRNA+confers+a+translational+advantage+that+can+be+achieved+in+cells+with+impaired+cap-dependent+translation.">The 5&#8217;-poly(A) leader of poxvirus mRNA confers a translational advantage that can be achieved in cells with impaired cap-dependent translation.</a> PLOS Pathogens. 13, e1006602 (2017).</li><li>Jha, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-kingdom+mimicry+underlies+ribosome+customization+by+a+poxvirus+kinase.">Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase.</a> Nature. 546, 651-655 (2017).</li><li>Dai, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+Profiling+Reveals+Translational+Upregulation+of+Cellular+Oxidative+Phosphorylation+mRNAs+during+Vaccinia+Virus-Induced+Host+Shutoff.">Ribosome Profiling Reveals Translational Upregulation of Cellular Oxidative Phosphorylation mRNAs during Vaccinia Virus-Induced Host Shutoff.</a> Journal of Virology. 91, e01858-e01816 (2017).</li><li>De Silva, F. S., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin-independent+plasmid+replication+occurs+in+vaccinia+virus+cytoplasmic+factories+and+requires+all+five+known+poxvirus+replication+factors.">Origin-independent plasmid replication occurs in vaccinia virus cytoplasmic factories and requires all five known poxvirus replication factors.</a> Journal of Virology. 2, 23 (2005).</li><li>Yang, Z., Bruno, D. P., Martens, C. A., Porcella, S. 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F., Moss, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-Wide+Analysis+of+the+5&#8242;+and+3&#8242;+Ends+of+Vaccinia+Virus+Early+mRNAs+Delineates+Regulatory+Sequences+of+Annotated+and+Anomalous+Transcripts.">Genome-Wide Analysis of the 5&#8242; and 3&#8242; Ends of Vaccinia Virus Early mRNAs Delineates Regulatory Sequences of Annotated and Anomalous Transcripts.</a> Journal of Virology. 85, 5897-5909 (2011).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+Chain+Reaction:+Basic+Protocol+Plus+Troubleshooting+and+Optimization+Strategies.">Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting and Optimization Strategies.</a> Journal of Visualized Experiments. , (2012).</li><li>Dieffenbach, C. W., Lowe, T. M., Dveksler, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+concepts+for+PCR+primer+design.">General concepts for PCR primer design.</a> PCR Methods and Applications. 3, S30-S37 (1993).</li><li>Innis, M. A., Gelfand, D. H., Sninsky, J. J., White, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> PCR Protocols: A Guide to Methods and Applications. , (2012).</li><li>Baklanov, M. M., Golikova, L. N., Malygin, E. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+on+DNA+Transcription+of+Nucleotide+Sequences+Upstream+to+T7+Promoter.">Effect on DNA Transcription of Nucleotide Sequences Upstream to T7 Promoter.</a> Nucleic Acids Research. 24, 3659-3660 (1996).</li><li>Thornton, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splicing+by+Overlap+Extension+PCR+to+Obtain+Hybrid+DNA+Products.">Splicing by Overlap Extension PCR to Obtain Hybrid DNA Products.</a> The Genetic Manipulation of Staphylococci: Methods and Protocols. , 43-49 (2017).</li><li>Akichika, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cap-specific+terminal+N6-methylation+of+RNA+by+an+RNA+polymerase+II&#8211;associated+methyltransferase.">Cap-specific terminal N6-methylation of RNA by an RNA polymerase II&#8211;associated methyltransferase.</a> Science. , (2018).</li><li>Sun, H., Zhang, M., Li, K., Bai, D., Yi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cap-specific,+terminal+N6-methylation+by+a+mammalian+m6Am+methyltransferase.">Cap-specific, terminal N6-methylation by a mammalian m6Am methyltransferase.</a> Cell Research. 1, (2018).</li><li>Boulias, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+the+m6Am+methyltransferase+PCIF1+reveals+the+location+and+functions+of+m6Am+in+the+transcriptome.">Identification of the m6Am methyltransferase PCIF1 reveals the location and functions of m6Am in the transcriptome.</a> bioRxiv. , 485862 (2018).</li><li>Dominissini, D., Rechavi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N4-acetylation+of+Cytidine+in+mRNA+by+NAT10+Regulates+Stability+and+Translation.">N4-acetylation of Cytidine in mRNA by NAT10 Regulates Stability and Translation.</a> Cell. 175, 1725-1727 (2018).</li><li>Wei, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+m6A,+m6Am,+and+m1A+Demethylation+Mediated+by+FTO+in+the+Cell+Nucleus+and+Cytoplasm.">Differential m6A, m6Am, and m1A Demethylation Mediated by FTO in the Cell Nucleus and Cytoplasm.</a> Molecular Cell. 71, 973-985 (2018).</li><li>Fu, Y., Dominissini, D., Rechavi, G., He, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+regulation+mediated+through+reversible+m6A+RNA+methylation.">Gene expression regulation mediated through reversible m6A RNA methylation.</a> Nature Reviews Genetics. 15, 293-306 (2014).</li></ol>

The authors would like to declare no competing financial interest.

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&#39;-UTR-AUG-) in T7 promoter added before the 5&#39;-UTR sequence. Although the transcription start site (TSS) starts from the first G at the 5&#39; end, decreasing the number of G&#39;s less than three in T7 promoter region decreased the...

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 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 synthesis6,7,8. To efficiently synthesize viral proteins, viruses evolved many strategies to hijack cellular translational machinery to redirect it for translation of viral mRNAs7,8. 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 mechanisms12,13,14. Over 100 poxvirus mRNAs have an evolutionarily conserved cis-acting element in the 5&#8217;-untranslated region (5&#8217;-UTR): a 5&#8217;-poly(A) leader at the very 5&#8217; ends of these mRNAs15,16. The lengths of these 5&#8217;-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&#8217;-poly(A) leader confers a translation advantage to an mRNA in cells infected with vaccinia virus (VACV), the prototypic member of poxviruses19,20.
The in vitro transcribed RNA-based luciferase reporter assay was initially developed to understand the role of 5&#8217;-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 in poxvirus-infected cells. First, plasmids are able to replicate in VACV-infected cells22. Second, cryptic transcription often occurs from plasmid DNA18,23,24. 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&#8217; to 3&#8217; direction: T7-Promoter, poly(A) leader or desired 5&#8217;-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&#8217;-UTR, 3&#8217;-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.

<table>
	<tbody>
		<tr>
			<td>2X-Q5 Master mix</td>
			<td>New England Biolabs</td>
			<td>M0492</td>
			<td>High-Fidelity DNA Polymerase used in PCR</td>
		</tr>
		<tr>
			<td>3&#180;-O-Me-m7G(5&#39;)ppp(5&#39;)G RNA Cap Structure Analog</td>
			<td>New England Biolabs</td>
			<td>S1411L</td>
			<td>Anti reverse Cap analog or ARCA</td>
		</tr>
		<tr>
			<td>Corning 96 Well Half-Area white flat bottom polystyrene microplate</td>
			<td>Corning</td>
			<td>3693</td>
			<td>Opaque walled 96 well white plate with solid bottom</td>
		</tr>
		<tr>
			<td>Dual-Luciferase Reporter Assay System</td>
			<td>Promega</td>
			<td>E1960</td>
			<td>Dual-Luciferase Assay Kit (DLAK)</td>
		</tr>
		<tr>
			<td>E.Z.N.A. Cycle Pure Kit</td>
			<td>OMEGA BIO-TEK</td>
			<td>D6492</td>
			<td>PCR purification kit</td>
		</tr>
		<tr>
			<td>GloMax Navigator Microplate Luminometer</td>
			<td>Promega</td>
			<td>GM2010</td>
			<td>Referred as multimode plate reader luminometer</td>
		</tr>
		<tr>
			<td>HiScribe T7 Quick High Yield RNA synthesis Kit</td>
			<td>New England Biolabs</td>
			<td>E2050S</td>
			<td>In-Vitro transcription kit</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Thermo Fisher Scientific</td>
			<td>11668019</td>
			<td>Cationic lipid transfection reagent</td>
		</tr>
		<tr>
			<td>NanoDrop2000</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-2000</td>
			<td>Used to measure DNA and RNA concentration</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985070</td>
			<td>Reduced serum media</td>
		</tr>
		<tr>
			<td>Purelink RNA Mini Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>12183018A</td>
			<td>RNA purification kit</td>
		</tr>
		<tr>
			<td>Vaccinia Capping System</td>
			<td>New England Biolabs</td>
			<td>M2080</td>
			<td>Capping system</td>
		</tr>
	</tbody>
</table>

in vitro transcribed rna-based luciferase reporter assay to study translation regulation in poxvirus-infected cells

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To dissect the role of 5&#39;-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&#39;-UTR and 3&#39;-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.

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...

Watch this Scientific Journal Video about In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells at JoVE.com

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

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&#8217;-untranslated region (UTR) and 3&#8217;-UTR. Different translation initiation modes can also be examined using this method.

Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5&#39;-poly(A) leader in the 5&#39;-UTR. To ...

Our protocol for an in vitro transcribed RNA-based luciferase assay helps study translation regulation in pox virus infected cells. Additionally, a customized length of the Poly(A)liter allows studying its regulatory effects on translation. The main advantage of this technique is that it allows for the study of translation regulation by Cis elements such as 5'UTR and 3'UTR in mRNA and broad spectrum of translation initiation.The in vitro transcribed RNA-based luciferase reporter assay may be tailored to incorporate modification to cap and other modification and used to test the effect on translation. To begin, design forward and reverse primers to generate PCR amplicon containing the following elements in 5'to 3'direction:T7-Promoter, Poly(A)liter, Firefly Luciferase ORF in a Poly(A)tail referred to as T7_12A-Fluc, include several extra nucleotides in forward primer followed by T7-Promoter Poly(A)liter or desired 5'UTR sequence and approximately 20 nucleotides corresponding to the 5'end of the reporter gene's ORF. Adjust the primer length based on melting temperature corresponding to the 5'end of the reporter gene's ORF ensuring that the corresponding region in the primer is identical to the sense strand of the gene.Then design reverse primer to include Poly(A)tail and approximately 20 nucleotides. Adjust based on melting temperature corresponding to the 3'end of the reporter gene's ORF ensuring that the corresponding region of the primer is identical to the antisense strand of the gene and an in-frame stop codon is present before the Poly(A)tail. 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 to as T7_Kozak-Rluc.To set up the reaction, add reagents in the following order to a PCR tube:DNase free water, 2X high-fidelity DNA polymerase, primers and sequence confirmed luciferase template DNA. After a standard three-step PCR cycle to generate a DNA template, detect the PCR product by running 5-10%of the PCR reaction in a 1%agarose TAE gel electrophoresis along with a molecular weight standard. To determine the size of the PCR product, visualize the gel under a UV illuminator.After determining the correct size of the PCR product, purify it using a commercially available PCR purification kit using 100 microliters of nuclease free water to elute the DNA. Check the concentration of the purified DNA using a spectrophotometer and store it at minus 20 degrees Celsius for use for in vitro transcription immediately. To synthesize RNA from the PCR product, add the following reagents to a microcentrifuge tube:DNase-RNase free water, NTP buffer mix, cap analog, PCR product template, and T7-RNA polymerase mix from in vitro transcription kit.Mix thoroughly. Incubate at 37 degrees Celsius for two hours and then proceed to the purification of the synthesized RNA using an RNA purification kit. To check the purified RNA, heat RNA at 70 degrees for five minutes to remove secondary structure and run it on 1.5 agarose TBE gel.Then visualize the gel under a UV illuminator. Check the concentration of the purified RNA using a spectrophotometer. Aliquot it and store at minus 80 degrees Celsius.Seed HeLa cells in a 24 well plate to achieve 80-90%confluency the following day and incubate overnight in an incubator at 37 degrees Celsius with 5%carbon dioxide. Infect HeLa cells with Vaccinia virus at a multiplicity of infection of five and keep uninfected HeLa cells for comparison. Then incubate the plate at 37 degrees Celsius with 5%carbon dioxide for 10 to 12 hours.After desired hours post infection, mix 480 nanograms of 12A sequence bearing Firefly Luciferase mRNA and 20 nanograms of Kozak sequence bearing Renilla Luciferase mRNA in one microcentrifuge tube. In another microcentrifuge tube, add 1.1 microliters of cationic lipid transfection reagent. Add 55 microliters of reduced serum media to both tubes, mix and incubate at room temperature for five minutes.Then add 55 microliters cationic lipid transfection reagent containing reduced serum media to mRNA containing tube. Mix gently but thoroughly with a pipette and incubate at room temperature for 15 minutes. During the incubation, remove the cell culture medium from the cells and add 400 microliters of reduced serum medium per well.When incubation is completed, add 100 microliters of the mixture drop wise and evenly per well of the 24 well plate. Five hours post co-transfection with 12A Fluc and Kozak Rluc mRNA, remove the reduced serum medium from the cells and lyse the cells by adding 150 microliters 1X lysis buffer from the Luciferase assay kit capable of performing two reporter assays. After incubating for 10 minutes at room temperature, scrape the cells using rubber and sterile syringe and transfer to a microcentrifuge tube.To pellet cell debris, centrifuge the lysate at 12, 000 times g for 10 minutes at four degrees Celsius. Transfer 30 microliters of supernatant per well of an opaque walled 96 well white assay plate with a solid bottom. Use the Luciferase assay kit and a multi-mode plate reader luminometer to measure the dual luminescence using kinetics function as described in the manuscript.After exporting the luminescence reading data into a desirable file format, determine relative translation rate from 12A Fluc mRNA in uninfected and VACV infected HeLA cells by dividing Fluc value by internal control Rluc value. This assay was used to test the translation efficiency of an Fluc mRNA that contains a 5'Poly(A)liter in uninfected and VACV infected cells. Both uninfected and VACV infected cells were successfully co-transfected with Fluc and Rluc mRNA.Dividing Fluc by Rluc normalized the transfection efficiency in RNA stability in cells. 5'Poly(A)liter containing mRNA has a translational advantage during VACV infection compared to uninfected cells. This was not due to differential transfection efficiency or mRNA stability as the RNA level was similar in uninfected and VACV infected cells five hours post mRNA transfection.Take special care when designing primers as this can affect not only PCR reaction efficiency but also all downstream processes requiring the amplified DNA. This method is an important assay to quickly test the translation regulation by Cis elements. If possible, this method should be corroborated by other complementary experiments.Precaution should be taken while using Vaccinia virus and avoid exposure to chemicals such as ethidium bromide and phenol.

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Translation: RNA to Protein

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14.5K Views.  Kansas State University. Our protocol for an in vitro transcribed RNA-based luciferase assay helps study translation regulation in pox virus infected cells. Additionally, a customized length of the Poly(A)liter allows studying its regulatory effects on translation. The main advantage of this technique is that it allows for the study of translation regulation by Cis elements such as 5'UTR and 3'UTR in mRNA and broad spectrum of translation initiation.The in vitro transcribed RNA-based luciferase reporter assay ...