Name: artificial rna polymerase ii elongation complexes for dissecting co-transcriptional rna processing events
Uploaded: 2019-05-13T14:00:00
Description: Watch this Scientific Journal Video about Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events at JoVE.com

We thank S. Shuman for providing the mammalian capping enzyme cDNA. This work was supported in part by a grant to the Stowers Institute for Medical Research from the Helen Nelson Medical Research Fund at the Greater Kansas City Community Foundation.&#160;&#160;Original data underlying this manuscript can be accessed from the Stowers Original Data Repository at http://www.stowers.org/research/publications/libpb-1434.

1. Assembly of Artificial Elongation Complexes and Pol II Walking
<ol>
	<li>Immobilize 1 nmol of non-template DNA oligo containing a 3&#8217; biotin molecule on magnetic beads. 
	NOTE: The following steps can be done in advance to prepare for future experiments. All oligo sequences used in this protocol are provided in Table 1. RNA oligos are synthesized with 5&#8217;-triphosphate modifications.
	<ol>
		<li>Add 200 &#181;L of magnetic beads (10 mg/mL) to a low-protein binding 1.5 ...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+capping:+biological+functions+and+applications.">mRNA capping: biological functions and applications.</a> Nucleic Acids Research. 44 (16), 7511-7526 (2016).</li><li>Ghosh, A., Lima, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymology+of+RNA+cap+synthesis.">Enzymology of RNA cap synthesis.</a> Wiley Interdisciplinary Reviews. RNA. 1 (1), 152-172 (2010).</li><li>Moteki, S., Price, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+coupling+of+capping+and+transcription+of+mRNA.">Functional coupling of capping and transcription of mRNA.</a> Molecular Cell. 10, 599-609 (2002).</li><li>Conaway, J. W., Conaway, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RNA+polymerase+II+transcription+factor+shares+functional+properties+with+Escherichia+coli+sigma+70.">An RNA polymerase II transcription factor shares functional properties with Escherichia coli sigma 70.</a> Science. 248, 1550-1553 (1990).</li><li>Wang, D., 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=Structural+basis+of+transcription:+backtracked+RNA+polymerase+II+at+3.4+angstrom+resolution.">Structural basis of transcription: backtracked RNA polymerase II at 3.4 angstrom resolution.</a> Science. 324 (5931), 1203-1206 (2009).</li><li>Wang, 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=Molecular+basis+for+5-carboxycytosine+recognition+by+RNA+polymerase+II+elongation+complex.">Molecular basis for 5-carboxycytosine recognition by RNA polymerase II elongation complex.</a> Nature. 523 (7562), 621-625 (2015).</li><li>Martinez-Rucobo, F. W., 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=Molecular+Basis+of+Transcription-Coupled+Pre-mRNA+Capping.">Molecular Basis of Transcription-Coupled Pre-mRNA Capping.</a> Molecular Cell. 58 (6), 1079-1089 (2015).</li><li>Mandal, S. 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=Functional+interactions+of+RNA-capping+enzyme+with+factors+that+positively+and+negatively+regulate+promoter+escape+by+RNA+polymerase+II.">Functional interactions of RNA-capping enzyme with factors that positively and negatively regulate promoter escape by RNA polymerase II.</a> Proceedings of the National Academy of Sciences USA. 101, 7572-7577 (2004).</li><li>Chiu, Y. 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=Tat+stimulates+cotranscriptional+capping+of+HIV+mRNA.">Tat stimulates cotranscriptional capping of HIV mRNA.</a> Molecular Cell. 10 (3), 585-597 (2002).</li><li>Vos, S. M., Farnung, L., Urlaub, H., Cramer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+paused+transcription+complex+Pol+II-DSIF-NELF.">Structure of paused transcription complex Pol II-DSIF-NELF.</a> Nature. 560 (7720), 601-606 (2018).</li><li>Vos, S. 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Studies that seek to dissect events coupled to the Pol II elongation complex such as RNA processing and regulation of the transcript elongation itself can be greatly facilitated by use of a highly purified enzyme system. Setting up such enzyme systems can be challenging. Promoter-dependent transcription by Pol II requires at least five general transcription factors. Preparing and stockpiling these factors can take months; hence, the rate-limiting step in this process is often simply preparing the cadre of transcription f...

Eukaryotic messenger RNA (mRNA) synthesis is an elaborate biochemical process that involves synthesis of an unprocessed precursor RNA by RNA polymerase II and processing of the precursor RNA to yield the mature mRNA. The RNA processing steps of capping, splicing, and polyadenylation are carried out largely co-transcriptionally. The Pol II elongation complex serves as a scaffold that recruits and orchestrates the activities of many of the RNA processing enzymes. Consequently, our ultimate understanding of how mature eukaryotic mRNAs are generated will rely heavily on the development of experimental systems to allow dissection of the biochemical mechanisms underlying recruitment to the elongation complex and regulation of enzymes responsible for co-transcriptional capping, splicing, and polyadenylation.
Not surprisingly, development of such experimental systems has been difficult. A major impediment has been the remarkable complexity of Pol II transcription itself where simply reconstituting basal transcription by Pol II in vitro requires a minimum set of five general transcription initiation factors: TFIIB, TFIID, TFIIE, TFIIF, and TFIIH1. Moreover, reconstituting any sort of regulated Pol II transcription in vitro requires an even larger set of transcription factors and coregulators. Thus, a major goal has been to develop simpler experimental systems allowing reconstitution of active Pol II elongation complexes suitable for investigations of the functional coupling of Pol II transcription and RNA processing.
One such simpler method for reconstituting active Pol II elongation complexes has proven useful for structural and biochemical studies of elongating Pol II and, more recently, for investigating co-transcriptional RNA processing2,3,4,5. In this article, we show how Pol II elongation complexes prepared from purified Pol II and synthetic transcription bubbles can be used effectively to investigate the mechanisms underlying co-transcriptional capping of nascent Pol II transcripts.
Capping refers to the covalent addition of a 5&#8217;-guanosine &#8220;cap&#8221; to the 5&#8217;-triphosphate end of nascent Pol II transcripts. The cap is important for subsequent steps of mRNA maturation, transport, translation, and other processes6,7. The cap is added co-transcriptionally to Pol II transcripts by an enzyme referred to as capping enzyme. In mammalian cells, active sites responsible for the RNA 5&#8217;-triphosphatase and guanylyl transferase activities of the capping enzyme are contained within a single polypeptide8. The capping enzyme is recruited to the Pol II elongation complex through interactions with yet to be defined surfaces on the Pol II body and the Rpb1 carboxy-terminal domain (CTD) phosphorylated on Ser5 of its heptapeptide repeats5. In the elongation complex, the capping enzyme catalyzes addition of a 5&#8217;-guanosine cap once the nascent transcript reaches a length of at least 18 nucleotides and has emerged from the polymerase RNA exit channel. In the first step of the capping reaction, the triphosphatase hydrolyzes the RNA 5&#8217;-triphosphate to yield a 5&#8217;-diphosphate. In the second step, GTP is hydrolyzed to GMP by the guanylyl transferase, forming a GMP-capping enzyme intermediate. Finally, the guanylyl transferase transfers GMP to the 5&#8217;-diphosphate end of the nascent transcript to produce the cap.
A remarkable feature of the capping reaction is that co-transcriptional capping (i.e., capping of transcripts associated with functional Pol II elongation complexes) is much more efficient than capping of free RNA5,9. Thus, a major question in the field has been how this dramatic activation of capping is achieved via interactions of the capping enzyme with the Pol II elongation complex. In this protocol we describe the assembly of active RNA polymerase II elongation complexes using only purified RNA polymerase II and artificial transcription bubbles. These methods allow creation of RNA polymerase II elongation complexes with transcripts of defined length and sequence. In a recent study, we used these defined RNA polymerase II elongation complexes as a model for investigating aspects of the mechanisms of RNA capping5. In particular, we showed that (i) capping of RNA associated with these elongation complexes was more than 100-fold more efficient than capping of free RNA and (ii) was stimulated by TFIIH-dependent phosphorylation of the Pol II CTD. The approach described here could in principle be adapted to generate substrates for studying other co-transcriptional RNA processing reactions linked to the Pol II elongation complex.
In Section 1 of this protocol, artificial elongation complexes are created by annealing a synthetic template strand DNA oligonucleotide to an RNA oligonucleotide that is complementary at its 3&#8217;-end to approximately 9 nucleotides of the template strand DNA. Pol II is then loaded onto the DNA:RNA duplex. The elongation complex is then completed by addition of a partially complementary, non-template strand DNA oligonucleotide that is labeled with biotin at its 3&#8217;-end (Figure 1 and Figure 2A). The RNA oligonucleotide is extended by Pol II in these elongation complexes to make radiolabeled transcripts of defined length and sequence upon addition of appropriate combinations of radiolabeled nucleotides. In addition, using a combination of washes to remove unincorporated nucleotides and further addition of different combinations of nucleotides, one can &#8220;walk&#8221; Pol II to different positions along the DNA template and synthesize RNA of defined lengths. RNA is then purified and subjected to electrophoresis in denaturing urea-PAGE gels. In Section 2 of the protocol, artificial elongation complexes are used to analyze co-transcriptional RNA capping. The example presented measures the effect of TFIIH-dependent phosphorylation of the Pol II CTD on co-transcriptional RNA capping. In this experiment, we measure the extent of co-transcriptional capping as a function of capping enzyme concentration (5, 15 and 45 ng per reaction) and time (1, 2 and 4 min).

<table>
	<tbody>
		<tr>
			<td>[&#945;-32P] UTP (3000 Ci/mmol), 1 mCi</td>
			<td>Perkin Elmer</td>
			<td>NEG007H001MC</td>
			<td>For radiolabeling RNA</td>
		</tr>
		<tr>
			<td>2x RNA loading dye</td>
			<td>New England Biolabs</td>
			<td>B0363S</td>
			<td>Highly recommended. For preparing RNA during gel loading</td>
		</tr>
		<tr>
			<td>40% Bis:Acrylamide solution</td>
			<td>Biorad</td>
			<td>1610144</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (20 mg/ml)</td>
			<td>New England Biolabs</td>
			<td>B9000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Cdk7/Cyclin H/MAT1 (CAK complex) Protein, active, 10 &#181;g</td>
			<td>Millipore Sigma</td>
			<td>14-476</td>
			<td>Used to phosphorylate Pol II CTD</td>
		</tr>
		<tr>
			<td>DNA oligonucleotides</td>
			<td>IDT</td>
			<td></td>
			<td>See Table 8 for purity specifications</td>
		</tr>
		<tr>
			<td>Dynabeads MyOne Streptavidin C1</td>
			<td>Life Technologies Invitrogen</td>
			<td>65001</td>
			<td>We have also used Dynabeads M-280 streptavidin without problem but prefer MyOne Streptavidin beads because they sediment more slowly</td>
		</tr>
		<tr>
			<td>GlycoBlue Coprecipitant (15&#8201;mg/ml)</td>
			<td>Life Technologies Invitrogen</td>
			<td>AM9516</td>
			<td>Highly recommended. Dyes nucleic-acid pellet blue making reactions much easier to handle</td>
		</tr>
		<tr>
			<td>Hoefer SE600 standard dual cooled vertical electrophoresis system with 1 mm spacers and 15 well comb</td>
			<td>Hoefer</td>
			<td>SE600-15-1.5</td>
			<td></td>
		</tr>
		<tr>
			<td>MaXtract high density tubes (1.5 ml)</td>
			<td>Qiagen</td>
			<td>129046</td>
			<td>Highly recommended. Contains a high density gel that forms a stable barrier between aqueous and organic phases; improves RNA yields during extractions</td>
		</tr>
		<tr>
			<td>Proteinase K solution (20&#8201;mg/ml)</td>
			<td>Life Technologies Invitrogen</td>
			<td>25530049</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA oligonucleotides</td>
			<td>Trilink</td>
			<td></td>
			<td>See Table 8 for more details</td>
		</tr>
		<tr>
			<td>Yeast Inorganic Pyrophosphatase (100 units/ml)</td>
			<td>New England Biolabs</td>
			<td>NEBM2403S</td>
			<td>Required only during capping reactions</td>
		</tr>
	</tbody>
</table>

artificial rna polymerase ii elongation complexes for dissecting co-transcriptional rna processing events

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3&#8217;-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility.
In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

Figures 2 and 3 show representative result reactions used to generate artificial elongation complexes containing transcripts of different lengths by extending or Pol II from different sources. Figure 4 depicts how these elongation complexes can be used to assay co-transcriptional CTD phosphorylation-dependent RNA capping.
Figure 2A is a...

Watch this Scientific Journal Video about Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events at JoVE.com

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

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