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

Summary

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.

Abstract

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’-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.

Introduction

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 re....

Protocol

1. Assembly of Artificial Elongation Complexes and Pol II Walking

1. Immobilize 1 nmol of non-template DNA oligo containing a 3’ 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’-triphosphate modifications.
   1. Add 200 µL of magnetic beads (10 mg/mL) to a low-protein binding 1.5 mL tube, and then place on a magnetic rack for 2 min.
   2. While the tube is on the magnetic rack, remove the liquid from the tube without distu....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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