This work was supported by the Department of Defense DoD grant AZ180075. Stefan Stamm thanks Jacqueline Noonan Endowment. Anna Pawluchin was supported by the DAAD, German academic exchange program, Justin R. Welden was a recipient of the University of Kentucky Max Steckler Award.

1. Design of the constructs
<ol>
	<li>Use the UCSC genome browser24 to identify repetitive elements necessary for circular RNA formation and incorporate them in the constructs. Importantly, primers for amplification need to be outside the repetitive elements.</li>
	<li>Paste the circular RNA sequence (Supplemental Figure 1 is a test sequence) into <a href="https://genome.ucsc.edu/cgi-bin/hgBlat?command=start" target="_blank">https://genome.ucsc.edu/cgi-bin/hgBlat?command=start</...

<ol>
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B., 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=Natural+RNA+circles+function+as+efficient+microRNA+sponges.">Natural RNA circles function as efficient microRNA sponges.</a> Nature. 495 (7441), 384-388 (2013).</li><li>Kelly, S., Greenman, C., Cook, P. R., Papantonis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exon+Skipping+Is+Correlated+with+Exon+Circularization.">Exon Skipping Is Correlated with Exon Circularization.</a> Journal of Molecular Biology. 427 (15), 2414-2417 (2015).</li><li>Li, Z., 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=Exon-intron+circular+RNAs+regulate+transcription+in+the+nucleus.">Exon-intron circular RNAs regulate transcription in the nucleus.</a> Nature Structural &amp; Molecular Biology. 22 (3), 256-264 (2015).</li><li>Yang, Y., 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=Extensive+translation+of+circular+RNAs+driven+by+N(6)-methyladenosine.">Extensive translation of circular RNAs driven by N(6)-methyladenosine.</a> Cell Research. 27 (6), 626-641 (2017).</li><li>Abe, N., 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=Rolling+Circle+Translation+of+Circular+RNA+in+Living+Human+Cells.">Rolling Circle Translation of Circular RNA in Living Human Cells.</a> Scientific Reports. 5, 16435 (2015).</li><li>Salzman, J., Chen, R. 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Nothing to disclose.

In general, circular RNAs are low abundant1, which complicates the study of their function and formation. Similar to linear RNAs13, the use of reporter minigenes allows the identification of cis and trans-acting factors that regulate the formation of circular RNAs. Thus, this approach generates hypotheses that can be further tested using the endogenous genes.
The most critical step is the design of the reporter gene. The enzymatic assembly of DNA...

Circular RNAs 
Circular RNAs (circRNAs) are covalently closed single stranded RNAs that are expressed in most organisms. They are generated by joining a downstream 5&#39; splice site to an upstream 3&#39; splice site, a process called back-splicing (Figure 1A)1. Sequences in the pre-mRNA that exhibit base complementary as short as 30-40 nt bring back-splice sites into proper alignment for circRNA formation2. In humans, Alu elements1, representing about 11% of the genome3, form extensive double stranded RNA structures in pre-mRNA due to their self-complementarity4,5 and thus promote the formation of circRNAs1.
Currently, three major functions of circRNAs have been described. Some circRNAs bind microRNAs (miRNAs) and through sequestration act like miRNA sponges6. CircRNAs have been implicated in transcriptional and post transcriptional regulation, through competition with linear splicing7 or modulation of transcription factor activity8. Finally, circRNAs contain short open reading frames and proof of principle studies show that they can be translated9,10. However, the function of most circRNAs remains enigmatic. The majority of circular RNAs have been detected using next-generation sequencing methods11. Detailed analyses of individual genes using targeted RT-PCR approaches reveal that a large number of circular RNAs remains to be discovered12.
Use of reporter genes to analyze pre-mRNA processing 
The analysis of mRNA derived from DNA reporter constructs transfected into cells is a well-established method to study alternative pre-mRNA splicing, which can be applied to circular RNAs. In general, the alternative exon, its surrounding introns, and constitutive exons are amplified and cloned into a eukaryotic expression vector. Frequently, the introns are shortened. The constructs are transfected into eukaryotic cells and usually analyzed by RT-PCR13,14. This approach has been extensively used to map regulatory splice sites and trans-acting factors in co-transfection experiments13,15,16,17,18. In addition, the generation of protein-expressing minigenes allowed for screening of substances that change alternative splicing19,20.
The method has been applied to circular RNAs. Currently, at least 12 minigene backbones have been described in the literature and are summarized in Table 1. With the exception of the tRNA based expression system21,22, they are all dependent on polymerase II promoters. Here, we describe a method to generate human reporter minigenes to determine cis and trans-acting factors involved in the generation of circular RNAs. An overview of the method using sequences of a published reporter gene23 is shown in Figure 1.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(PEI) Hydrochloride</td>
			<td>Polysciences</td>
			<td>24765-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Builder tool</td>
			<td>NEB</td>
			<td></td>
			<td><a href="https://nebuilder.neb.com/#!/" target="_blank">https://nebuilder.neb.com/#!/</a></td>
		</tr>
		<tr>
			<td>Dark Reader Transilluminator.</td>
			<td>Clare Chemical Research</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Enzymatic DNA assembly kit</td>
			<td>NEB</td>
			<td>E2621S</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel and PCR cleanup kit</td>
			<td>Promega</td>
			<td>A9282</td>
			<td></td>
		</tr>
		<tr>
			<td>Glyco Blue</td>
			<td>Thermo Fisher</td>
			<td>AM9516</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3.1 cloning site</td>
			<td>Polycloning site</td>
			<td></td>
			<td><a href="https://assets.thermofisher.com/TFS-Assets/LSG/manuals/pcdna3_1_man.pdf" target="_blank">https://assets.thermofisher.com/TFS-Assets/LSG/manuals/pcdna3_1_man.pdf</a></td>
		</tr>
		<tr>
			<td>Polymerase 1</td>
			<td>NEB</td>
			<td>M0491L</td>
			<td>Q5 DNA polymerase</td>
		</tr>
		<tr>
			<td>Polymerase 2</td>
			<td>Biorad</td>
			<td>1725310</td>
			<td>Long range polymerase (NEB), iproof (BioRad)</td>
		</tr>
		<tr>
			<td>Polymerase 2</td>
			<td>Qiagen</td>
			<td>206402</td>
			<td>Qiagen long range polymerase kit</td>
		</tr>
		<tr>
			<td>Reverse Transcriptase</td>
			<td>Thermo Fisher</td>
			<td>18080044</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA isolation kit</td>
			<td>Life Technologies</td>
			<td>12183025</td>
			<td>Ambion by Life Technologies</td>
		</tr>
		<tr>
			<td>RNAse R</td>
			<td>Lucigen</td>
			<td>RNR07250</td>
			<td>Epicenter/Lucigen</td>
		</tr>
		<tr>
			<td>Stable competent cells</td>
			<td>NEB</td>
			<td>C3040H</td>
			<td>NEB stable cells</td>
		</tr>
		<tr>
			<td>Standard cloning bacteria</td>
			<td>NEB</td>
			<td>C2988J</td>
			<td>NEB5-alpha competent</td>
		</tr>
		<tr>
			<td>Web tool to design primers</td>
			<td>NEB</td>
			<td></td>
			<td><a href="https://nebuilder.neb.com/#!/" target="_blank">https://nebuilder.neb.com/#!/</a></td>
		</tr>
		<tr>
			<td>Web-based temperature calculations</td>
			<td>NEB</td>
			<td></td>
			<td><a href="https://tmcalculator.neb.com/#!/main" target="_blank">https://tmcalculator.neb.com/#!/main</a></td>
		</tr>
	</tbody>
</table>

use of alu element containing minigenes to analyze circular rnas

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an upstream 3&#39; splice site within a pre-mRNA, a process called back-splicing. This circularization is likely aided by secondary structures in the pre-mRNA that bring the splice sites into close proximity. In human genes, Alu elements are thought to promote these secondary RNA structures, as Alu elements are abundant and exhibit base complementarities with each other when present in opposite directions in the pre-mRNA. Here, we describe the generation and analysis of large, Alu element containing reporter genes that form circular RNAs. Through optimization of cloning protocols, reporter genes with up to 20 kb insert length can be generated. Their analysis in co-transfection experiments allows the identification of regulatory factors. Thus, this method can identify RNA sequences and cellular components involved in circular RNA formation.

Reporter genes allow determination of regulatory factors that influence circular RNA formation. However, these reporter genes are large and contain repetitive elements that often make DNA constructs unstable. Due to their large size, it is often necessary to delete parts of the introns, which is achieved by amplifying genomic pieces containing the exons and smaller flanking intronic parts. These DNA pieces are enzymatically assembled, allowing construction without restriction enzymes.
The exam...

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Use of Alu Element Containing Minigenes to Analyze Circular RNAs

We clone and analyze reporter genes generating circular RNAs. These reporter genes are larger than constructs to analyze linear splicing and contain Alu elements. To investigate the circular RNAs, the constructs are transfected into cells and resulting RNA is analyzed using RT-PCR after removal of linear RNA.

Use of Alu Element Containing Minigenes to Analyze Circular RNAs

In addition to linear mRNAs, many eukaryotic genes generate circular RNAs. Most circular RNAs are generated by joining a 5&#39; splice site with an ...

This protocol is significant because it allows the user to generate a genomic expression system that can undergo alternative splicing and in this case form circular RNAs that can be tested for their function and disease association. The main advantage of this technique is that it will pave the way for others to use this protocol in molecular biology and cloning when studying alternative splicing in circular RNA formation and function. My advice to someone trying this technique for the first time would be to optimize the PCR conditions.Run gradients or perform touchdown PCR with different annealing temperatures and extension times. Usually longer fragments over six KB will extend one KB per cycle and different annealing temperatures would need to be tested for the best amplification. Demonstration of this method is critical because it will give the users a better visual representation of the more difficult aspects of the procedure especially the generation of primers.To begin this procedure, load the UCSC Genome Browser and use it to identify repetitive elements necessary for circular RNA formation and incorporate them into the constructs. Importantly, primers for amplification need to be outside the repetitive elements. Paste the circular RNA sequence into the human BLAT search and select the right organism.Submit the sequence, go to browser view, and zoom out 1.5 timers or as appropriate. Next, mouse over the repetitive elements to identify their subtype in a floating window. Alu elements are in the sine line.Use the default tracks button under the window to reset the browser if an incorrect image is obtained. First go to view, DNA on the top line of the USCS Genome Browser to download the DNA sequence shown in the window. In the sequencing formatting option, select extended case/color options.Select the default case as lower and select toggle case for NCBI RefSeq. Select underline and bold and Italic for RepeatMasker. Click submit.There will be exons as capital letters and introns as lowercase letters. Check the exon/intron borders. If the browser shows the reverse complement, go back and select the reverse complement box until the correct exon/intron borders are show.Next, copy the file with the correct orientation into a word processing document and highlight the exons. Select the fragments to be amplified making sure that the intron does not begin or end in a repetitive region as primers in these regions will not amplify specific sequences. To begin, use a web tool to design the primers for cloning.For the vector sequence, add the insertion site as the last nucleotide and subsequently add the fragments. Since the vector numbering does not start with a given insertion site, the site of insertion in the vector is located and the downstream part is put in in front of the upstream sequence. Adjust primers if their melting points are more than four degrees Celsius apart and do not work in amplification.In this study, reporter genes that generate circular RNAs are cloned and analyzed. Optimized PCR products are separated on a 1%agarose gel containing 1X gel green. The individual bands represent the PCR products that will be used in enzymatic DNA assembly.These bands are then cut out from the gel and purified. The purified PCR product does not run true to the expected size with gel green so the products are also separated on a 1%agarose gel that is subsequently stained with ethidium bromide to ensure the products are the correct size. To begin, set out an enzymatic DNA assembly kit.Combine the vector and inserts in a molar ratio of one to two. Next, add 10 microliters of DNA assembly master mix. Incubate samples for 60 minutes at 50 degrees.Thaw competent cells on ice during incubation. Cells should be in a volume of 50 microliters. Next, transform competent cells with the total assembly reaction.Add two microliters of the chilled assembled product to the competent cells. Mix by gently flicking the tube four to five times. Do not vortex.Place the mixture on ice for 30 minutes. Heat shock the mixture at 42 degrees Celsius for 30 seconds. Then place it back on ice for two minutes.After this, add 950 microliters of room temperature SOC media to the tube. Incubate at 37 degrees Celsius for 60 minutes while shaking at 300 RPM. During this incubation, warm two selection plates that contain the appropriate antibiotic.Following the incubation, centrifuge the reaction tube at 10, 000 g for 30 seconds to pellet the cells and plate out 25%of the cells on one selection plate and 75%on the other. Incubate these plates overnight at 37 degrees Celsius. Before you begin transfection, check your DNA by restriction digest.Here, a representative tau minigene that contains exons nine through 12 is cut with restriction enzymes indicated to rule out major recombinations. For transfection, first dissolve linear polyethylene hydrochloride in water at a concentration of one milligram per milliliter at pH two. Use sodium hydroxide to bring the pH up to seven and sterile filter the solution with a 0.22 micrometer filter.Store the solution at four degrees Celsius until ready to use. Then split the cells into the wells of a six-well plate and let them grow overnight at DMEM media containing 10%FBS. The next day, aliquot one microgram of the reported gene in a sterile tube and add 200 microliters of sterile filtered 150 millimolar sodium chloride.Mix by vortexing. Next, add the polyethylenimine solution to this mixture at a ratio of three microliters of polyethylenimine for each one microgram of DNA. Centrifuge briefly to collect samples at the bottom of the tube.Incubate the samples at room temperature for 10 minutes. Then add it directly to HEK293 cells. Incubate the cells overnight at 37 degrees Celsius with 5%carbon dioxide.The next day, use an RNA isolation kit to isolate the RNA for RT-PCR. cDNA from two samples derived from human brain tissues are amplified with circular RNA primers circ tau exon 1210 reverse and circ tau exon 1211 forward. While the expected band corresponding to tau circular RNA is seen, the other strong bands are artifacts that did not match the human genome.This experiment is repeated with identical PCR conditions, but the reverse transcription was performed only with the circ tau exon 1210 reverse primer. Only the expected band is amplified and validated through sequencing. The RNA is then treated with RNase R that removes linear RNA.The circular RNA is detectable after the treatment whereas linear RNA gives no longer a detectable signal. RNA is isolated 24 hours post-transfection and analyzed by RT-PCR. Amplification of the linear tau mRNA shows two bands due to alternative splicing of exon 10.Their ratio changes to the over expression of splicing factors. Amplification of the circular 1210 tau RNA shows the dependency of tau circ RNA expression on the expression of some splicing factors especially the Cdc2-like kinase CLK2 and the SR protein 9G8. The most important thing to remember when attempting this procedure is the design and location of the primers when constructing a minigene.The primer should not lie in repetitive elements and will need to be optimized under different conditions depending on the fragment being amplified. Following this procedure, other methods that can be performed will be testing the function of the circular RNAs. The users can test translation or sequestration of proteins to identify a function for the specific circular RNA the user is interested in.This technique paved the way to utilize genomic fragments in a minigene system that can over express the circular RNAs allowing the user to test and identify their function and in some aspects their association to disease. The implication of this technique extends towards potential therapies and diagnostics of a particular disease, for instance tauopathies, neurodegenerative diseases associated with tau pathology. We have discovered that the microtubule-associated protein tau, known as MAPT, generates circular RNAs which we believe are contributing to the disease pathology and this method allows us to fully study these circular RNAs and identifying their function and relation to disease.This method could provide insight into a better understanding of circular RNAs, what their functions are, and the role they may play in certain diseases. This method can be applied in cloning other minigenes that express circular RNAs across species where it can provide insight into their function. Amplification of the PCR products proves to be the most difficult with long fragments amplified from genomic DNA, along with having multiple repetitive elements within the fragment.

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JoVE Journal

Biochemistry

7.2K vistas.  University of Kentucky. Este protocolo es significativo porque permite al usuario generar un sistema de expresión genómica que puede someterse a empalmes alternativos y en este caso formar ARN circulares que se pueden probar para su función y asociación de enfermedad. La principal ventaja de esta técnica es que allanará el camino para que otros utilicen este protocolo en biología molecular y clonación al estudiar el empalme alternativo en la formación y función circulares de ARN. Mi consejo a alguien que p...