RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Summary

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

Abstract

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based “mix and measure” hammerhead ribozyme in vitro reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro RNA editing or high throughput screening of chemicals that can inhibit the editosome function.

Introduction

The process of RNA editing, a post-transcriptional mRNA modification, was first discovered in trypanosomatids¹. Since then, substantial work has been conducted in studying the mechanism behind RNA editing in Trypanosoma brucei^2,3. In a series of enzymatic reactions, the editosome, a core complex of about 20 proteins, creates mature mitochondrial mRNAs for multiple components of the energy generating oxidative phosphorylation system. The order of catalytic events is endonucleolytic cleavage, uridylate (U) addition or deletion, and ligation, as dictated by guide RNAs (gRNAs)⁴.

In addition to the core editosome complex proteins, a number of accessory factors have also been identified^5-7. These proteins are mostly seen grouped in independent complexes. However, the order of protein assembly in the core editosome complex and the interaction patterns of the core complex with the accessory complexes are yet to be determined. Targeting the RNA editing process in trypanosomatids may provide chemical dissectors that aid in studying the assembly and function of the editosome complex. Furthermore, functional studies on several editosome proteins have shown essentiality across different life stages, indicating their potential as drug targets^8-12. Therefore, the found inhibitors of the editosome may also act as lead compounds against trypanosomatids. This is timely, as drugs currently available against diseases caused by trypanosomatid are toxic, inefficient and expensive^13,14.

An efficient and convenient in vitro assay is necessary to explore the chemical universe for specific inhibitors that block RNA editing. Three assays have been developed and used to monitor editosome activities: (a) full round in vitro RNA editing assay¹⁵, (b) pre-cleaved in vitro RNA editing assay^16,17, and (c) hammerhead ribozyme (HHR)-based assay¹⁸. The first two assays rely on direct visualization of the edited product (ATPase 6 mRNA) with the help of radioactivity. The HHR-based assay uses a modified version of the ATPase 6 mRNA that is modeled to behave as a ribozyme upon editing. The functional ribozyme then specifically cleaves a radiolabeled RNA substrate, serving as a reporter. Recently, Moshiri et al. developed a ‘mix and measure’ HHR-based in vitro reporter assay to monitor RNA editing where the radiolabeled RNA substrate is replaced with a fluorescence resonance energy transfer (FRET) substrate¹⁹. The principle advantages of this assay are: (a) it is a rapid and convenient mix and measure type of assay, as the production of active ribozyme and substrate cleavage occur simultaneously in the same tube in low volume (i.e. 20 μl), (b) it avoids the use of radioactively labeled materials, (c) sensitivity that is afforded by fluorescence instrumentation in a micro-titer plate format, and (d) a high signal to noise ratio. Using this assay, the effect of known RNA editing ligase inhibitors against purified editosome was confirmed¹⁹. This experiment validated the assay for rapid identification of RNA editing inhibitors, primarily against whole editosomes from T. brucei.

Figure 1 is a detailed step-by-step schematic of the fluorescence-based in vitro RNA editing assay. This protocol can either be used for monitoring RNA editing in vitro or easily be adapted for screening compound libraries of various scales.

Protocol

The protocol below describes the procedure for performing the fluorescence-based RNA editing assay. The assay can be performed in a single PCR tube, 96-well, or 384-well plates depending on the scope of the experiment. Subsequently the fluorescence signal can be read on a suitable real time PCR detection system. The assay here is described in the context of 384-well plates.

1. Culturing T. brucei Cells

1. Prepare a growth medium for T. brucei procyclic form cells. For 1 L of medium:
   1. Dissolve 25.4 g SDM-79 powder in 800 ml miliQ water.
   2. Add 2 g of NaHCO₃ and pH to 7.3 with 10 M NaOH.
   3. Add nanopure water to a final volume of 900 ml, filter sterilize.
   4. Add Fetal Bovine Serum (FBS), penicillin-streptomycin solution and hemin (2.5 mg/ml) to final concentrations of 10% (v/v), 100 U/ml and 7.5 mg/L respectively.
2. Grow 300 ml of T. brucei 1.7A wild type (procyclic form) cells at 28 °C, shaking at 70 rpm to a density of 1.5 x 10⁷ cells/ml. NOTE: This should produce 3 ml of active editosome with ~0.5 mg of total protein, sufficient for 600 editing reactions.
3. Harvest the cells by centrifugation at 6,000 x g for 10 min at 4 °C.
4. Wash the pellet with 50 ml of chilled PBSG buffer (10 mM Na₂HPO₄, 10 mM NaH₂PO₄, 145 mM NaCl, and 6 mM glucose), and spin down the cells again by centrifugation at 10,000 x g for 10 min at 4 °C.

2. Isolation of Crude Mitochondria

NOTE: All the steps should be performed on ice or at 4 °C to preserve editosome activity.

1. Resuspend the harvested cells in 30 ml of DTE buffer (1 mM Tris-HCl pH 8.0 and 1 mM EDTA). Use a 40 ml sterile Dounce homogenizer (pre-chilled) to disrupt the cell membrane by stroking up and down at least 10 times on ice.
2. Immediately add 4.3 ml of 60% sucrose (w/v; i.e. 1.75 M) to the homogenate to a final concentration of 0.25 M. Centrifuge at 15,800 x g for 10 min at 4 °C, to preferentially bring down mitochondria.
3. Resuspend the mitochondrial pellet in 4.6 ml of STM buffer (20 mM Tris-HCl pH 8.0, 250 mM sucrose and 2 mM MgCl₂). Add 13.8 μl of 0.1 M CaCl₂ and 4 μl of RNase-free DNase I to final concentrations of 0.3 mM and 9 U/ml, respectively. Incubate the mixture for 1 hr on ice.
4. Add 4.6 ml of STE buffer (20 mM Tris-HCl pH 8.0, 250 mM sucrose and 2 mM EDTA) to inactivate the DNase I. Centrifuge at 15,800 x g for 10 min at 4 °C.
5. Resuspend the pellet in 400 μl of lysis buffer (10 mM Tris-HCl pH 7.2, 10 mM MgCl₂, 100 mM KCl, 1 µg/ml pepstatin, 1 mM DTT, and 1x complete EDTA-free protease inhibitor) and transfer to a microfuge tube.
6. Add 10% Triton X-100 to a final concentration of 1% and incubate the lysate for 15 min at 4 °C on a tube rotator.
7. Clear the mitochondrial lysate by centrifuging twice at 17,000 x g for 15 min at 4 °C; retaining the cleared supernatant each time.

3. Editosome Purification

1. Pour a 10 ml 10%-30% (v/v) linear glycerol gradient (Table 1) in an ultracentrifuge tube using 2x HHE gradient buffer (40 mM HEPES pH 7.9, 20 mM Mg(OAc)₂, 100 mM KCl, and 2 mM EDTA) and a gradient maker by following the instruction manual.
2. Carefully remove 500 μl of solution from the top of the glycerol gradient and gently load 500 μl of the cleared mitochondrial lysate. Spin at 178,000 x g for 6 hr at 4 °C using an ultracentrifuge.
3. Collect 500 µl fractions sequentially from the top to the bottom of the gradient at 4 °C. Then snap freeze the fractions using liquid nitrogen and store at -80 °C until usage.

4. RNA Preparation

1. Anneal the respective DNA template containing sequence complementary to T7 promoter sequence (Table 2) with a T7 promoter oligonucleotide (5’-TAATACGACTCACTATAGGG-3’) in a 1:1 molar ratio by heating at 90 °C for 3 min and cooling at RT for at least 10 min.
2. Transcribe RNA using an in vitro transcription kit by following the instruction manual.
3. Stop the transcription reaction by adding equal volume of 7 M urea dye (7 M urea, 0.05% Xylene Cynol, and 0.05% Bromophenol blue). Run on a filter sterilized 9% denaturing polyacrylamide gel (9% acrylamide, 7 M urea, 1x TBE) .
4. Use the ultraviolet (UV) shadowing with a shortwave UV lamp to locate and excise respective RNA. Place the excised gel piece in a microfuge tube and add 400 µl of gel elution buffer (20 mM Tris-HCl pH 7.5, 250 mM NaOAc, 1 mM EDTA and 0.25% SDS). Elute overnight at RT on a tube rotator.
5. Precipitate the eluted RNA by adding 1 ml of cold 100% ethanol and incubating either at -80 °C for 30 min or -20 °C overnight.
6. Centrifuge at 16,000 x g for 30 min at 4 °C, to pellet down the RNA.
7. Wash the pellet with 1 ml of 75% ethanol. Centrifuge at 16,000 x g for 20 min at 4 °C.
8. Resuspend the RNA pellet appropriately in RNase free water to achieve the desired concentrations, as shown in Table 2.

5. Fluorescence-based RNA Editing Assay

1. For a single reaction, combine 1 pmol (1 µl) of preA6Rbz and 2.5 pmol (1 µl) of gA6Rbz (1:2.5 molar ratio) in a microfuge tube, incubate at 70 ºC for 3 min and let it sit at RT for at least 10 min.
2. Meanwhile prepare a master mix using Table 3, without the preA6Rbz and gA6Rbz, for the editing reaction containing 1x HHE buffer (25 mM HEPES pH 7.9, 10 mM Mg(OAc)₂, 50 mM KCl and 10 mM EDTA), 1 mM ATP, 5 mM CaCl₂, 16 ng/µl of Torula yeast RNA, 0.1% Triton X-100, and the purified editosome.
3. Add annealed preA6Rbz and gA6Rbz to complete the master mix.
4. Dispense 18 µl of the master mix (Table 3) into wells containing either 2 µl of RNase-free water (wells with no compounds) or 2 µl of 200 µM chemical compounds and include control samples in the plate according to Figure 5.
5. Seal the plate with a plate sealer and spin the plate down, to remove any air bubbles. Incubate at 28 °C for 4 hr.
6. Add 25 pmol (2 µl) of gA6Rbz competitor to each well. Place a fresh sealer, spin the plate down and place it on a real-time PCR machine. Program the following experiment:
   Step 1: 85 °C for 5 min; Step 2: 24 °C for 10 min; Step 3: Stop.
7. Add 15 pmol (1 µl) of FRET substrate to each well to a final volume of 23 µl. Seal the plate with a fresh sealer. Quickly spin the plate and place it back on the real-time PCR machine.
8. Program a new experiment with the following steps:
   Step 1: 37 °C for 1 min; Step 2: Read; Step 3: Go to Step 1, 40 times; Step 4: Stop.
9. Setup the plate by selecting all the wells that require reading and choose the FAM filter. Input volume as 23 μl and start the run.
10. Calculate the slope of the values obtained from each well/sample to obtain a kinetic measurement by plotting the slopes on a bar graph for analysis. NOTE: A kinetic read improves the signal-to-noise ratio between the sample and the background; as the background sample would have a slope close to zero. An end point reading has higher background noise.

Results

To demonstrate the necessary steps required for setting up a large-scale screen, Figures 2-5 are representative control experiments related to the quality of the assay. These are essential control experiments for a consistent assay over several days of screening or for comparison of different screens.

Assessing the Fluorescence Signal-to-noise Ratio

To ensure the stability and quality of the fluorescein-labeled oligoribonucleo...

Discussion

A novel high-throughput screening method to identify inhibitors against the RNA editing complex of Trypanosomes was presented, providing a new tool for drug discovery to counter diseases caused by trypanosomatids. FRET-based ribozyme assay has been extensively used for different purposes^20-22; however, we have utilized the capacity of FRET-based ribozyme assay for in vitro monitoring of RNA editing activity¹⁹. This assay could potentially be adapted to other types of RNA editing in eukaryot...

Materials

Name	Company	Catalog Number	Comments
SDM-79 Medium	Gibco by Life Technologies
Fetal Bovine Serum	Life Technologies	12483-020	heat inactivation at 55 °C for 1 hr
Hemin, minimum 80%	Sigma	H5533-10G
Penicillin-Streptomycin Sollution	Fisher Scientific	MT-30-002-CI
Dnase 1 recombinant, Rnase Free	Roche	4716728001
T7 RiboMax Express Large  scale RNA  production system	Promega	P1320
Kimble Kontes Dounce Tissue Grinders	Fisher Scientific	K885300-0040
Gradient Master, ver 5.25	Biocomp	107-201M
Ultra Clear Tube, 13.2 ml	Beckman Coulter	344059
Optima L-100XP  Ultracentrifuge	Beckman Coulter	392052
SW 41 Ti ROTOR	Beckman Coulter	331336
MicroSeal 'B' Seal, Seals	Biorad	MSB1001
CFX 384 Touch Real-Time PCR Detection System	Biorad	185-5484
Acryl/Bis solution (19:1), 40% (w/v)	Bio Basic	A0006-500ML
Urea, Molecular biology grade, 1 kg	Life Technologies	AM9902