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 chan....

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....

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.