Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

Summary

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

Abstract

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.

We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

Introduction

Gene expression profiling is a key tool used to study cellular processes and the associated complex interaction network. Studies on mRNA abundance have typically been the method of choice to obtain basic insights into the underlying molecular mechanisms. The development of whole-transcriptome microarrays ¹ and, more recently, next-generation sequencing of RNA (RNA-seq) ^2-4 fueled this approach. While these technologies have revolutionized our understanding of the complexity of cellular gene expression, they face major limitations due to intrinsic properties of their template sample, i.e. total cellular RNA. First, short-term changes in total RNA levels do not match changes in transcription rates, but are inherently dependent on the RNA half-life of the respective transcripts. While a fivefold induction of a short-lived transcript, e.g. encoding for a transcription factor, will be readily detectable in total RNA within an hour, the same induction of a long-lived transcript, e.g. encoding for a metabolic enzyme, will remain virtually invisible. In addition, even a complete shut-down (>1,000-fold down-regulation) in the transcription rate of an average gene with an RNA half-life of five hours will simply take five hours for its total RNA levels to decrease by only twofold. Therefore, analysis of total RNA favors the detection of up-regulation of short-lived transcripts, many of which encode for transcription factors and genes with regulatory functions ⁵. In addition, the true kinetic cascade of regulation is obscured and primary signaling events cannot be differentiated from secondary. Both, in turn, may result in substantial bias in downstream bioinformatics analyses. Second, alterations in total RNA levels cannot be attributed to changes in RNA synthesis or decay. Measurements of the latter require cell invasive approaches, e.g. blocking transcription using actinomycin D ⁶, and extended monitoring of ongoing RNA decay over time. With a mean mRNA half-life in mammalian cells of 5 - 10 hr ^5,7, mRNA levels of most genes will only have decreased by less than twofold following several hours of transcriptional arrest. These rather small differences result in grossly imprecise measurements of mRNA half-lives for the majority of cellular genes due to the exponential nature of the underlying mathematical equations. Finally, while RNA-seq of total cellular RNA revealed that approximately half of our genes are subject to alternative splicing events⁸, the underlying kinetics as well as the dynamic mechanisms guiding tissue- and context-specific regulation of RNA processing remain poorly understood. In addition, the contribution of RNA processing to differential gene expression, particularly for non-coding RNAs, remains to be determined. Altogether, these limitations represent major obstacles for bioinformatic kinetic modeling of the underlying molecular mechanisms.

We recently developed an approach, termed high resolution gene expression profiling, to overcome these problems ^5,7,9. It is based on metabolic labeling of newly transcribed RNA using 4-thiouridine (4sU-tagging), a naturally occurring uridine derivative, and provides direct access to newly transcribed transcripts with minimal interference in cell growth and gene expression (see Figure 1) ^5,10-12. Exposure of eukaryotic cells to 4sU results in its rapid uptake, phosphorylation to 4sU-triphosphate, and incorporation into newly transcribed RNA. Following isolation of total cellular RNA, the 4sU-labeled RNA fraction is thiol-specifically biotinylated generating a disulfide bond between biotin and the newly transcribed RNA. 'Total cellular RNA' can then be quantitatively separated into labeled ('newly transcribed') and unlabeled ('pre-existing') RNA with high purity using streptavidin-coated magnetic beads. Finally, labeled RNA is recovered from the beads by simply adding a reducing agent (e.g. dithiothreitol) cleaving the disulfide bond and releasing the newly transcribed RNA from the beads.

Newly transcribed RNA depicts the transcriptional activity of every gene during the timeframe of 4sU exposure. 4sU-tagging in the timescale of minutes thus provides a snapshot picture of eukaryotic gene expression and an ideal template for down-stream bioinformatic analyses (e.g. promoter analysis). In cases where steady-state conditions can be assumed, the ratios of newly transcribed/total, newly transcribed/unlabeled and unlabeled/total RNA provide non-invasive access to precise RNA half-lives ^7,13. In addition, it is important to note that newly transcribed RNA purified after as little as 5 min of 4sU-tagging (5 min 4sU-RNA) is younger than 15 and 60 min 4sU-RNA. When performing both ultra-short and progressively longer 4sU-tagging in a single experimental setting combined with RNA-seq, the kinetics of RNA processing are revealed at nucleotide resolution ⁹. Finally, time-course analyses of newly transcribed and total RNA combined with computational modeling allow an integrative analysis of RNA synthesis and decay ¹⁴.

In conclusion, this approach allows for the direct analysis of the dynamics of RNA synthesis, processing, and degradation in eukaryotic cells. It is applicable in all major model organisms including mammals, insects (Drosophila), amphibians (Xenopus), and yeast ^5,15,16. It is directly compatible with microarray analysis ^5,17, RNA-seq ^9,13,14, and is applicable in vivo^12,15. Here, we detail the methodology to label, isolate, and purify newly transcribed RNA in cultured mammalian cells. In addition, potential problems and pitfalls are discussed.

Protocol

1. Metabolic Labeling with 4-thiouridine

Make a detailed plan of the experimental setup/schedule, e.g. when to add the 4sU to cell culture and when to harvest the samples. Plan for at least 5 min in between each condition. Only treat cells of one condition at a time. Handle max. 3 - 5 dishes at a given time. Handle cells as quickly as possible to minimize changes in temperature and CO₂ levels. Avoid exposing the cells to bright light after 4sU is added as this may result in crosslinking of 4sU-labeled RNA to cellular proteins.

Start of labeling

1. Thaw 4-thiouridine (4sU) just before use and pipette required amount of 4sU for each condition into a sterile Falcon tube.
2. Take the required amount of cell culture medium (5 ml per 10 cm dish) off the dishes and add to 4sU-containing Falcon tube and mix thoroughly. Remove and discard the remaining medium from the dishes.
3. Apply 4sU-containing medium back to the dishes.

End of labeling

4. Remove cell culture medium from cells. Add 5 ml of Trizol to each plate. For complex experiments including multiple time points or conditions, this step is best done by two people, one removing the medium, the other adding Trizol and harvesting the lysate.
5. Incubate for 5 min at room temperature for complete cell lysis.
6. Use a 10 ml pipette to rinse the plate carefully with the added Trizol. This aids complete cell lysis and sample recovery. Handle with care as Trizol is extremely hazardous when getting in contact with skin or eyes! Have antidote for phenol burns at hand (e.g. Polyethylene glycol 300 or 400 in industrial methylated spirits (70:30)). Transfer samples to polypropylene tubes. Please note that standard Falcon tubes do not resist these high g forces). Samples can be stored at -20 °C for at least one month until total RNA is prepared.

2. RNA Preparation Using Modified Trizol Protocol

1. Add 1 ml chloroform (0.2 ml per ml Trizol) and shake vigorously for 15 sec. Incubate at room temperature for 2 - 3 min.
2. Centrifuge at 13,000 × g for 15 min at 4 °C.
3. Transfer aqueous upper phase (containing the RNA) to a new 15 ml polypropylene tube.
4. Add ½ the reaction volume of both RNA precipitation buffer and isopropanol (e.g. to 3 ml supernatant add 1.5 ml RNA precipitation buffer and 1.5 ml isopropanol).
5. Mix well. Incubate at room temperature for 10 min.
6. Centrifuge at 13,000 × g for 10 min at 4 °C. Discard supernatant.
7. Spin down briefly (5,000 × g for 30 sec) and remove residual isopropanol with 200 μl pipette.
8. Add an equal volume of 75% ethanol and shake tube until the pellet detaches. Avoid breaking it into many small pieces as this may make removal of residual ethanol difficult.
9. Centrifuge at 13,000 × g for 10 min at 4 °C. Discard supernatant.
10. Spin down RNA briefly and remove remaining ethanol with a 200 μl pipette. Repeat step and remove remaining ethanol with a 20 μl pipette. After these two steps, no further drying of the pellet should be performed.
11. Add 100 μl of H₂O per 100 μg expected RNA yield and mix well by pipetting up and down 5 - 6 times to aid in dissolving the RNA.
12. Dissolve and denature RNA by heating to 65 °C for 10 min (shaker) and immediately place on ice.
13. Measure RNA concentration at 260 nm using a NanoDrop spectrophotometer, following manufacturer's instructions. This RNA can be stored at -80 °C for at least one month.

3. Thiol-specific Biotinylation of Newly Transcribed RNA

1. Start with 60 - 80 μg of total cellular RNA.
2. Constitute labeling reaction. Pipette in the following order (per μg RNA):
   1. 1 μl 10x Biotinylation Buffer
   2. 7 μl RNA (containing 1 μg RNA diluted in nuclease-free H₂O)
   3. 2 μl biotin-HPDP (1 mg/ml DMF)

Always add the biotin-HPDP last and mix immediately by pipetting. In case the biotin precipitates, DMF content can be increased to a final concentration of 40%.

3. Incubate at room temperature for 1.5 hr with rotation.
4. Add an equal volume of chloroform. Mix vigorously. Incubate for 2 - 3 minutes until the phases begin to separate and bubbles start to disappear.
5. Centrifuge at 20,000 × g for 5 min at 4 °C. Carefully transfer the upper aqueous phase into a new tube.
6. Repeat steps 3.4 and 3.5 once. You may want to perform this step in 2 ml Phase Lock Gel Heavy tubes to reduce loss of RNA.
7. RNA precipitation: add 1/10 the volume of 5 M NaCl and an equal volume of isopropanol to the water phase.
8. Centrifuge at 20,000 × g for 20 min at 4 °C. Discard supernatant.
9. Add an equal volume of 75% ethanol, centrifuge at 20,000 × g for 10 min at 4 °C, discard supernatant.
10. Spin briefly and remove residual ethanol with 200 μl pipette.
11. Spin briefly and remove residual ethanol with 20 μl pipette.
12. Do not allow RNA to dry. Re-suspend it in 50 - 100 μl H₂O (~1 μl per 1 μg input RNA). Mix well by pipetting up and down 5 - 6 times.
13. Check RNA quality by electrophoretical analysis to exclude RNA degradation.

4. Dot Blot Analysis of 4sU-incorporation (Optional)

4sU incorporation can be readily determined by dot blot analysis of biotinylated RNA. This is an optional step that allows trouble shooting and estimation of 4sU incorporation rates relative to a biotinylated DNA oligo control. For this assay we recommend using iodoacetyl-biotin instead of biotin-HPDP for biotinylation of 4sU-labeled RNA in step 3.2. This results in an irreversible biotinylation of 4sU-RNA. Therefore, column-based methods (e.g. RNeasy) can be used for recovery of much smaller amounts of biotinylated RNA (e.g. 5 μg). While RNA biotinylated using biotin-HPDP is also suitable for this assay, the resulting signal is weaker and the signal-noise ratio less favorable (Figure 3).

1. Follow the protocol for 4sU-labeling and isolation of total cellular RNA as described in sections 1 and 2.
2. Biotinylate 4sU-labeled RNA as described in section 3 replacing biotin-HPDP with iodoacetyl-biotin and perform two chloroform extractions to completely remove excessive iodoacetyl-biotin residues.
3. Recover biotinylated RNA by isopropanol/ethanol precipitation as described or using a column-based approach (e.g. RNeasy) in case small amounts of RNA (<10 μg) are used.
4. Incubate the Zeta membrane in nuclease-free water with rocking for 10 min.
5. Take the membrane out of the nuclease-free water and remove excessive fluids by placing membrane in between two clean paper towels and pressing firmly. Air-drying the membrane for 5 min will result in nicer dots.
6. For each sample, prepare 20 μl of 200 ng/μl RNA using ice cold dot blot binding buffer (10 mM NaOH, 1 mM EDTA). Apply 5 μl of this dilution (i.e. 1 μg of RNA) as well as three subsequent 10-fold dilutions (i.e. 100, 10, and 1 ng RNA, respectively) to the Zeta membrane by pipetting. Pipetting through an empty rack of pipette tips can be employed to provide evenly distributed spacing. Alternatively, use a dot blot apparatus according to the manufacturer's instructions.
7. Apply 5 μl of the biotin-labeled DNA oligo at concentrations ranging from 20 ng/μl to 20 pg/μl (i.e. 100 to 0.1 ng oligo) as a positive control to the membrane by pipetting. Use a biotinylated, 4sU-naive sample as negative control.
8. Air-dry the membrane for 5 min.
9. Incubate the membrane for 30 min in 40 ml blocking buffer with rocking.
10. Incubate the membrane with 10 ml of 1:1,000 streptavidin-horseradish peroxidase for 15 min (5 ml PBS + 5 ml 20% SDS + 10 μl streptavidin-horseradish peroxidase)
11. Wash membrane twice in 40 ml PBS + 10% SDS (20 ml PBS + 20 ml 20% SDS) for 5 min.
12. Wash membrane twice in 40 ml PBS + 1% SDS (38 ml PBS + 2 ml 20% SDS) for 5 min.
13. Wash membrane twice in 40 ml PBS + 0.1% SDS (40 ml PBS + 200 μl 20% SDS) for 5 min.
14. Remove excessive fluid by placing membrane in between two clean paper towels and pressing on them firmly.
15. Visualize membrane-bound HRP using ECL per manufacturer's instructions.
16. Place the membrane in plastic foil/bag, remove air bubbles and incubate for 2 min in the dark.
17. Expose membrane to film for 1 - 5 min.

5. Separation of Labeled and Unlabeled RNA Using Streptavidin-coated Magnetic Beads

1. Heat washing buffer (3 ml per sample) to 65 °C in a water bath.
2. Prepare fresh 100 mM dithiothreitol (DTT) in nuclease-free H₂O. Do so by decanting 15 - 30 mg of DTT powder into a clean 50 ml Falcon tube placed on the ultra-fine scale. Weigh and add required amount of nuclease-free H₂O.
3. Heat biotinylated RNA samples to 65 °C for 10 min to denature and immediately place on ice.
4. Place μMacs columns into the magnetic stand. We recommend not to process more than 12 samples at a time (6 - 8 samples are optimal).
5. Pre-equilibrate Miltenyi columns with 1 ml room temperature washing buffer. This will take about 15 min.
6. Meanwhile, add 100 μl of streptavidin beads to 50 - 100 μl of biotinylated RNA. Incubate at room temperature for 15 min with rotation.
7. If any of the columns has not initiated draining by now this can be facilitated by gently pressing on the top of the column with a gloved finger. Once the flow has started the columns drain readily.
8. Apply the RNA/beads to the columns. Discard the flow-through unless you want to recover the unlabeled RNA fraction (see section 7).
9. Wash three times with 0.9 ml of 65 °C washing buffer (1 ml pipette tips shrink when pipetting buffers at 65 °C).
10. Wash three times with 0.9 ml room temperature washing buffer.
11. Pipette 700 μl Buffer RLT (RNeasy MinElute Cleanup Kit, Qiagen) into new 2 ml tubes and place them underneath the columns.
12. Elute the newly transcribed RNA into the RLT Buffer by adding 100 μl of 100 mM DTT to the columns.
13. Perform a second elution round 3 min later into the same tube by adding another 100 μl of 100 mM DTT.

6. Recovery of Newly Transcribed RNA

Continue with the RNeasy MinElute Cleanup (Qiagen) protocol following the manufacturer's instructions. Elute in 25 μl nuclease-free H₂O. Measure RNA concentrations using a Nanodrop Spectrophotometer. To avoid the need to thaw and re-freeze RNA before submitting it to a high-throughput assay, we recommend preparing cDNA immediately after the newly transcribed RNA is purified. Use 2.5 μl of the newly transcribed RNA in 20 μl cDNA synthesis mix for cDNA synthesis following the manufacturer's instructions. Perform qRT-PCR controls using 1:10 dilutions of the cDNA mix. Store RNA at -80 °C.

7. Recovery of Unlabeled, Unbound RNA (Optional)

In case the unbound RNA needs to be recovered; collect and combine the flow-through (after adding the RNA-streptavidin beads solution to the columns) and the first wash for subsequent precipitation. Usually it is sufficient to precipitate only 50% of the unbound RNA as this will contain >80% of the starting material.

1. Add an equal volume of isopropanol (no salt needs to be added as the washing buffer already contains 1 M NaCl).
2. Centrifuge at 20,000 × g for 20 min at 4 °C. Discard supernatant.
3. Add an equal volume of 75% ethanol, centrifuge at 20,000 × g for 10 min at 4 °C, discard supernatant.
4. Spin briefly and remove residual ethanol with 200 μl pipette.
5. Spin briefly and remove residual ethanol with 20 μl pipette.
6. Do not allow RNA to dry. Resuspend it in 100 μl H₂O. Mix well by pipetting up and down 5 - 6 times. Incubate at 65 °C for 10 min with shaking and transfer directly to ice.
7. Check RNA quality by electrophoretical analysis to exclude RNA degradation.

Results

1. Starting Material and Expected Yields

Following 1 hour (hr) of 4sU-exposure newly transcribed RNA represents about 1 - 4% of total cellular RNA. This will be lower in growth-arrested cells as they no longer synthesize RNA to account for cell growth/replication. When labeling for 1 hr, we recommend starting the assay with 60 - 80 μg of total RNA. Starting with less than 30 μg of total RNA results in small RNA pellets that are hard to see after the biotinylation step and thus may be easily...

Discussion

Metabolic labeling of newly transcribed RNA substantially enhances the power of high-throughput technologies like microarrays and RNA-seq by providing more suitable templates to address the biological question of interest. The present protocol underwent extensive optimization. It allows >1,000-fold enrichment of newly transcribed RNA and provides highly reproducible results.

The experimental design of a 4sU-tagging experiment is of crucial importance as newly transcribed RNA will depi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4-thiouridine	Carbosynth	T4509	Prepare 50 mM stock in sterile H2O, store at -20 °C in aliquots of 50-500 μl, discard unused reagent, do not refreeze.
Trizol	Invitrogen	15596026 (100 ml), 15596018 (200 ml)	WARNING - CORROSIVE and HAZARDOUS TO HEALTH! Ensure immediate access to Phenol antidote (PEG-Methanol); Store at 4 °C.
Chloroform	Sigma	372978	WARNING - HAZARDOUS TO HEALTH
Isopropanol	Sigma	650447
Sodium citrate, nuclease-free	Sigma	C8532	Prepare 1.6 M stock solution using nuclease-free water.
5M nuclease-free NaCl	Sigma	71386	Stock solution
Nuclease-free H2O	Sigma	W4502	Make 1 ml aliquots in nuclease-free tubes.
RNA precipitation buffer			1.2 M NaCl, 0.8 M sodium citrate in nuclease free water. Prepare in advance under strictly nuclease-free conditions. Store at room temperature in 50 ml falcon tubes.
Ethanol	Sigma	459844	Use with nuclease-free water to prepare 80% ethanol, store at -20 °C.
1 M nuclease-free Tris Cl, pH 7.5	Lonza	51237	Stock solution
500 mM nuclease-free EDTA, pH 8.0	Invitrogen	15575-020	Stock solution
10x Biotinylation Buffer (BB)			100 mM Tris pH 7.4, 10 mM EDTA in nuclease-free water, make aliquots of 1 ml.
Dimethylformamide (DMF)	Sigma	D4551
EZ-Link biotin-HPDP	Pierce	21341	Prepare 1 mg/ml stock solution by dissolving 50 mg biotin-HPDP in 50 ml DMF. Gentle warming enhances solubilisation. Store at 4 °C in aliquots of 1 ml.
Phase Lock Gel Heavy tubes 2.0 ml	Eppendorf	0032 005.152	Optional for the chloroform extraction step.
Zeta membrane	BIORAD	162-0153
10x Dot blot binding buffer			100 mM NaOH, 10 mM EDTA
Biotin-oligo			5'-biotin, 25 nucleotides, any sequence
Sodium dodecyl sulphate	Fisher	BPE9738	For 100 ml 20% stock solution, add 20 g SDS to 80 ml PBS pH 7-8 and adjust volume to 100 ml. Keep all high-percentage SDS solutions above 20 °C. Warm the solutions slightly should SDS precipitate.
EZ-Link Iodoacetyl-LC-Biotin	Pierce	21333	Prepare 1 mg/ml stock solution by dissolving 50 mg iodoacetyl-biotin in 50 ml DMF. Gentle warming enhances solubilisation. Store at 4 °C in aliquots of 1 ml. Generates irreversible, thiol-specific biotinylation.
Phosphate buffer saline	Gibco	10010-015
Dot blot blocking buffer			Mix 20 ml 20% SDS with 20 ml 1 x PBS pH 7-8 and add EDTA to the final concentration of 1 mM.
Streptavidin-horseradish peroxidase	Vector Laboratories	SA5004	Store at -20 °C. Mix 10 ml 20% SDS with 10 ml 1 x PBS. Add 20 μl Streptavidin-HRP before use.
ECL reagent	GE Healthcare	RNP2109	Use following the manufacturer's instructions.
Super RX, X-RA Film, 18x24 cm	Fujifilm	47410 19236
μMacs Streptavidin Kit	Miltenyi	130-074-101	Store the beads at 4 °C.
Tween 20	Sigma	P1379
Washing buffer			100 mM Tris pH 7.4, 10 mM EDTA, 1 M NaCl, 0.1% Tween 20 in nuclease-free H2O.
Dithiothreitol (DTT)	Sigma	43817	Prepare as 100 mM DTT in nuclease-free H2O, always prepare fresh before use.
RNeasy MinElute Kit	Qiagen	74204	Store columns at 4 °C, remaining components of the kit at room temperature.
1.5 ml screw-top polypropylene tubes	Sarstedt	72.692.005	Compatible with Dimethylformamide
2.0 ml screw-top polypropylene tubes	Sarstedt	72.694.005	Compatible with Dimethylformamide
15 ml tubes	BD Falcon	352096	Compatible with Dimethylformamide
50 ml tubes	BD Falcon	352070	Compatible with Dimethylformamide
All solutions/reagents should be stored at room temperature unless otherwise specified.
Equipment
UV/VIS spectrophotometer	Thermo Scientific	NanoDrop 1000	Or equivalent. Use low volume (1-2 μl) for measurements of low RNA concentrations to avoid excessive sample loss.
Polypropylene 15 ml centrifuge tubes	VWR International	525-0153	In contrast to standard 15 ml tubes, these tolerate up to 15,000 × g
High-speed centrifuge	Beckman Coulter	Avanti J-25	Or equivalent equipment capable of reaching 13,000×g
High-speed rotor	Beckman Coulter	JLA-16250	Or equivalent equipment capable of reaching 13,000×g
Adaptors for 15 ml tubes	Laborgeräte Beranek	356964	Or equivalent equipment capable of reaching 13,000×g
Refrigerated table-top centrifuge	Eppendorf	5430 R	Or equivalent.
Thermomixer	Eppendorf	Thermomixer compact	Or equivalent.
Magnetic stand	Miltenyi Biotec	130-042-109	One stand holds 8 μMacs columns.
Waterbath	Grant	SUB Aqua 5	Or equivalent.
Ultra-fine scale	A&D	GR-202	Or equivalent.
E-Gel iBase Power System	Invitrogen	G6400UK	For RNA gels; or equivalent.
E-Gel EX 1% agarose precast gels	Invitrogen	G4020-01	For RNA gels; or equivalent.