Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation

Summary

This method can be used to measure RNA synthesis. 5-Bromouridine is added to cells and incorporated into synthesized RNA. RNA synthesis is measured by RNA extraction immediately after labelling, followed by 5-Bromouridine-targeted immunoprecipitation of labelled RNA and analysis by reverse transcription and quantitative polymerase chain reaction.

Abstract

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in production or degradation of RNA. This protocol describes a method for measurement of RNA production, using 5-Bromouridine labelling of RNA followed by immunoprecipitation, which enables investigation of RNA synthesized within a short timeframe (e.g., 1 h). The advantage of 5-Bromouridine-labelling and immunoprecipitation over the use of toxic transcriptional inhibitors, such as α-amanitin and actinomycin D, is that there are no or very low effects on cell viability during short-term use. However, because 5-Bromouridine-immunoprecipitation only captures RNA produced within the short labelling time, slowly produced as well as rapidly degraded RNA can be difficult to measure by this method. The 5-Bromouridine-labelled RNA captured by 5-Bromouridine-immunoprecipitation can be analyzed by reverse transcription, quantitative polymerase chain reaction, and next generation sequencing. All types of RNA can be investigated, and the method is not limited to measuring mRNA as is presented in this example.

Introduction

5-Bromouridine (BrU) immunoprecipitation (IP) allows the study of RNA production in cells with no or very limited effects on cell physiology during the brief labelling period^1,2. The method is based upon incorporation of the synthetic uridine derivative BrU into newly synthesized RNA followed by IP of labelled RNA using anti-BrU antibodies (Figure 1).

It has been known for decades that protein synthesis can be transcriptionally regulated, and the existence of transcription factors was hypothesized more than 50 years ago³. Today, it is known that several diseases are caused by dysregulation of transcription and RNA stability (Supplementary Figure S1 in reference⁴), and the ability to measure changes in RNA production is of great importance in understanding disease development.

On the other hand, tools to regulate RNA production provide new possibilities for treating diseases caused by too little or too much protein expression, or protein accumulation such as in Parkinson's disease (PD). The predominant protein accumulating as insoluble aggregates in PD is α-synuclein, and the level of α-synuclein is directly linked to the disease, as gene multiplication of the α-synuclein gene causes familial PD⁵. Furthermore, α-synuclein aggregates in a concentration dependent manner. Downregulation of α-synuclein mRNA levels is therefore an interesting therapeutic strategy, which has been successfully achieved using RNA interference to decrease neuronal cell loss in PD rodent models^6,7.

It is important to be able to measure the changes in RNA production caused by either disease or therapeutic interventions. State of the art methods for measuring steady state levels of RNA, such as reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR), are not capable of distinguishing between changes caused by transcriptional regulation or by altered RNA stability. A widely used method for investigation of RNA decay rates is blocking of the transcriptional machinery using compounds such as α-amanitin or actinomycin D followed by measurements of the decaying RNAs. However, there are some problems associated with an overall blockage of transcription in cells, such as induction of apoptosis^8,9. Beside the cytotoxic effects, transcriptional inhibitors also present several technical issues, such as slow cellular uptake of α-amanitin and lack of specificity of actinomycin D (reviewed in reference¹⁰).

To avoid the overall blockage of transcription, nucleotide analogues have been used, such as 5-ethynyl uridine (5-EU), 4-thiouridine (4-TU), and BrU. These are readily taken up by mammalian cells and incorporated into newly synthesized RNA, enabling pulse-labelling of RNA within a specific time-frame. BrU is less toxic than 5-EU and 4-TU, making it the preferred analogue of choice^11,12.

BrU-IP can be used to investigate both the rate of RNA synthesis and stability, and thus distinguish between the underlying causes of changes in total RNA. This article will focus on the measurement of RNA synthesis and refers to reference² for details on investigation of RNA stability. To investigate RNA synthesis, cells are briefly labelled with BrU e.g. for 1 h followed by BrU-IP¹ (Figure 1C). This enables measurements of RNA synthesized within the short labelling time and observed changes will give a better estimate of regulations in RNA synthesis than by measuring changes in total RNA by RT-qPCR. It should be mentioned, however, that even though the labelling time is, for example, only 1 h, degradation can still have an influence on the RNA levels observed.

RNA from BrU-IP experiments are suitable for downstream analysis by both RT-qPCR¹ or next generation sequencing^2,13. Other standard RNA detection methods, such as northern blotting or ribonuclease protection assay, may also be applicable for certain RNAs.

Protocol

NOTE: Perform all steps at room temperature unless otherwise stated and keep buffers on ice or in the fridge (except from the Elution buffer containing SDS). The protocol is divided into 5 sections: Preparation of RNA samples; preparation of beads for IP; binding of BrU-labelled RNA to beads; elution and purification of bound BrU-labelled RNA by 3 steps phenol-phenol/chloroform-chloroform extraction; analysis of RNA (in this example using RT-qPCR)

1. Preparation of RNA Samples

1. Count HEK293T cells using an automated cell counter and seed 3,500,000 cells in a 10 cm petri dish in 10 mL growth media (Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum and 50 µg/mL penicillin/streptomycin) to obtain a total of >40 µg RNA upon extraction 48 h later. Maintain cells at 37 °C and 5% CO₂.
2. 48 h after seeding, aspirate 8 mL growth media and leave 2 mL behind to avoid drying out the cells. Add 2 mM BrU and any treatment to the 8 mL aspirated growth media, and remove the residual 2 mL from the cells before reapplying the 8 mL BrU containing growth media. Avoid the use of fresh media upon application of the BrU to the cells, since this can induce changes in gene expression.
3. Incubate the cells for 1 h with BrU and treatment. Wash the cells in 5 mL Hank's buffer and trypsinize the cells using 2 mL 0.05% trypsin. Add 8 mL growth media to stop the reaction, transfer the cells to a 15 mL tube, and spin down the cells for 2 min at 1,000 x g.
4. Remove the supernatant and extract total RNA from the cell pellet using an RNA isolation kit (see Table of Materials) or any other preferred method, and elute in RNase-free H₂O. Store the RNA at -80 °C until ready to proceed.

2. Preparation of Beads for IP

1. Prepare beads in batches for the number of samples to be investigated plus one extra to account for loss during pipetting and whirl mixing. Resuspend anti-Mouse IgG magnetic beads thoroughly by whirl mixing and take out 20 µL beads per sample in an RNase-free 1.5 mL tube. Place the 1.5 mL tube in a magnetic stand and leave approximately 20 s for the beads to collect on the side.
2. Remove the supernatant, take out the tube from the magnetic stand, and resuspend in 400 µL 1x BrU-IP buffer by pipetting. Spin the tube very briefly for 2 s in a table-top centrifuge, place it back in the magnetic stand, and remove the supernatant. Repeat the washing step once.
3. Block unspecific binding to the beads using heparin. Resuspend the beads in 1 mL 1x BrU-IP+ 1 mg/mL heparin buffer, and leave rotating for 30 min. Wash the beads once, as in step 2.2.
   NOTE: Heparin is a negatively charged polymer, which will compete with RNAs for binding. tRNA or other irrelevant RNAs can also be used if the downstream application is not sequencing.
4. Resuspend the beads in 1 mL 1x BrU-IP buffer and add 1.25 µg/sample anti-α-Bromodeoxyuridine antibody, which also recognizes BrU. Incubate the beads for 1 h while rotating, or overnight at room temperature.
5. Wash the beads three times as in step 2.2. Resuspend the beads in 50 µL/sample 1x BrU-IP buffer supplemented with 1 mM BrU and leave rotating for 30 min to lower the sensitivity of the antibody bound to the beads and hereby the risk of unspecific binding during IP.
6. Wash the beads three times as in step 2.2, and resuspend in 50 µL/sample 1xBrU-IP buffer.

3. Binding of BrU-Labelled RNA to Beads

1. Measure RNA concentrations in the RNA extracts from step 1.3 using ultraviolet-visible spectrophotometry, and dilute 40 µg RNA from each sample in RNase-free H₂O to a total volume of 200 µL.
2. Heat RNA samples for 2 min at 80 °C to denature RNA and spin briefly in a tabletop centrifuge. Add 200 µL 2x BrU-IP+BSA/RNAse inhibitor.
3. Add 50 µL resuspended and prepared beads from step 2.6 to each sample and leave rotating 1 h. Wash the beads four times as in step 2.2.

4. Elution and Purification of RNA by 3 Steps Phenol-phenol/Chloroform-chloroform Extraction

1. Elute RNA and perform phenol extraction
   1. Resuspend the beads in 200 µL Elution buffer to elute RNA, and quickly thereafter add 200 µL phenol, pH 6.6 (CAUTION: Toxic, see note below step 4.1.2) to remove any non-RNA molecules from the sample.
   2. Whirl mix the sample, and spin 3 min at 25,000 x g in a tabletop centrifuge.
      NOTE: Phenol is toxic and should be handled in a fume hood wearing skin protection. The slightly acidic pH ensures that only RNA, and not DNA, is extracted. The RNA will remain in the upper aqueous phase due to the hydrophilic nature of charges. The hydrophobic cores of proteins will bind phenol and move to the lower organic phenol-phase.
2. Perform phenol-chloroform extraction
   1. Aspirate as much upper-phase as possible (approximately 190 µL of the 200 µL, depending on experience in handling) and move it to a tube with 200 µL phenol/chloroform, pH 6.6 (CAUTION: Toxic, see note). Make sure to aspirate the same amount across samples.
   2. Whirl mix the sample and spin for 3 min at 25,000 x g.
      NOTE: Chloroform is toxic and should be handled in a fume hood wearing skin protection. Chloroform is added to remove phenol, which can affect the downstream analysis of RNA by inhibiting reverse transcription¹⁴ and polymerase chain reactions¹⁵.
3. Perform chloroform extraction by aspirating the aqueous upper phase as before (now approximately 180 µL), and transfer to a tube containing 200 µL chloroform. Whirl mix the sample and spin 3 min at 25,000 x g.
4. Aspirate the aqueous upper phase as before (now approximately 170 µL) and transfer it to a tube containing 17 µL (1/10 volume) 3 M CH₃COONa, pH 6.0. To neutralize and precipitate RNA from the aqueous solution, add 2 µL glycogen (20 mg/mL), which precipitates together with RNA and makes the RNA pellet more visible.
5. Add 500 µL 96% ethanol to increase binding between the salt ions and RNA and mix by inverting the tube a couple of times. Leave the tube overnight at -20 °C or 15 min on dry ice.
6. Spin the sample at 25,000 x g, 4 °C for 30 min. Discard supernatant without disturbing the pellet and wash the pellet in 185 µL 75% ethanol to remove any residual salt. Spin at 25,000 x g, 4 °C for 5 min.
7. Discard supernatant completely without disturbing the pellet and leave the pellet to dry 5-10 min with an open lid. Resuspend in 10 µL RNase-free H₂O applied on the side of the tube to avoid disturbing the pellet.

5. Analysis of RNA

1. Prepare cDNA using a cDNA reverse transcription kit (see Table of Materials) or any other preferred method using 2 µL RNA sample and 1 µg yeast total RNA as carrier RNA for the cDNA synthesis reaction (not used if cDNA is used for next generation sequencing). Use 1 µg RNA without yeast RNA for cDNA preparation of corresponding input.
2. Dilute cDNA 1:25 and mix 9 µL diluted cDNA with 10 µL qPCR Master mix and 1 µL fluorogenic probe, both specified in the Table of Materials.
   NOTE: The cDNA dilution depends on the cDNA preparation and qPCR method used.
3. Run the following qPCR program (e.g., on a 7500 Fast Real-time PCR system or equivalent): 2 min at 50 °C, 20 s at 95 °C, 40 cycles of 3 s at 95 °C and 30 s at 60 °C.

Results

Our initial tests of BrU-labelling time were performed in AsPC-1 cells. The cells were treated with BrU for 0, 1, 2, and 4.5 h, followed by total RNA extraction and BrU-IP. GAS5 and GAPDH were measured in BrU-IP RNA, which demonstrated that 1 h labelling was sufficient to reach an approximately 9- and 44-fold change compared to background (0 h) for GAPDH and GAS5, respectively.

BrU-IP and the analysis described above were used t...

Discussion

This protocol describes the use of BrU-IP for determination of RNA production by labelling of newly produced RNA for 1 h with BrU, followed by immediate RNA extraction and immunoprecipitation of BrU-labelled RNA. This method has previously been described in reference¹⁶, and this article includes some additional steps to increase IP specificity and RNA purity, by pre-treating beads with low concentrations of BrU as well as a phenol-chloroform purification step to increase RNA purity following IP. A...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ribolock Rnase inhibitor	ThermoFisher	EO0381
Dynabeads M-280 Sheep anti-mouse IgG	Life Technologies	11202D
α-Bromodeoxyuridine mouse antibody	BD Bioscience	555627
Nanodrop 1000	Thermo Fisher		Ultraviolet-visible spectrophotometry
Water-Saturated Phenol pH 6.6	ThermoFisher	AM9712
Phenol:Chloroform:IAA 25:24:1 pH 6.6	ThermoFisher	AM9732
High Pure RNA isolation Kit	Roche	11828665001
High Capacity cDNA Reverse Transcription Kit	ThermoFisher	4368814
Taqman Fast Advanced Master Mix	ThermoFisher	4444557	qPCR Master Mix
Taqman RNA probes	ThermoFisher	SNCA: Hs01103383_m1, 18sRNA: Hs03928985_g1, ACTB: Hs01060665_g1, HMBS: Hs00609296, NADH: Hs01072843_m1	Flurogenic probes
7500 Fast Real-Time PCR system	Applied Biosystems
HEK293T cell line	ATCC
Dulbecco's Modified Eagle's Medium	Lonza	BE12-604F
Fetal calf serum	Biochrom	S0115
Penicillin/Streptomycin	Biochrom	A2213
Hank's buffer			5,3 mM KCl, 0.44 mM KH2PO4, 0.2 mM Na2HPO4 and 136.8 mM NaCl pH 6.77, autoclaved.
DEPC-H2O			0.1% diethylpyrocarbonate treated H2O
2xBrU-IP Buffer			40 mM Tris-HCl pH 7.5 and 500 mM NaCl in DEPC H2O
2xBrU-IP+BSA/RNAse inhibitor			2xBrU-IP buffer supplemented with 1 μg/μL BSA and 80 U/mL Ribolock
BrU-IP+ 1 mg/mL heparin			2xBrU-IP buffer diluted 1:1 DEPC-H2O and supplemented with 1mg/mL heparin
1xBrU-IP			2xBrU-IP buffer diluted 1:1 in DEPC-H2O and supplemented with 0.5 μg/μL BSA and 20 U/mL Ribolock
Elution buffer			0.1% SDS in RNAse-free H2O